KR20180134858A - Integrated cell - Google Patents

Abstract

The cell support material is prepared by providing an aqueous solution of silk protein that can be assembled into a water insoluble macrostructure. Silk proteins are mixed with eukaryotic cells, and silk proteins are assembled into water-insoluble macrostructures in the presence of cells to form support material for cell culture. The cells may grow integrated with the support material under conditions suitable for cell culture.

Description

Integrated cell

The present invention relates to the field of eukaryotic cell culture and tissue engineering, and provides a method for culturing eukaryotic cells and a cell scaffold material, wherein a silk protein such as fibroin or spider silk protein a polymer of silk protein is used as the cell support material.

The basic concept of tissue engineering is to combine different components such as living cells, biomaterials and bioactive factors to form engineered tissue structures. Traditional tissue engineering strategies typically employ a " top-down " approach wherein the cells are seeded onto a polymeric support. The material should then include large pores with high interconnectivity to allow subsequent infiltration. In order to have high porosity and not collapse, the material must have thick and / or stiff walls, which can cause poor cell compatibility and low flexibility when cells try to expand resulting in flexibility.

Alternatively, a " bottom-up " tissue engineering approach has recently begun. The bottom-up approach relies on assembling the matrix with cells and smaller components or modules. For example, this approach can be achieved by 3D printing of hydrogel containing cells. However, one major disadvantage of hydrogels is the lack of mechanical strength, which limits its use for soft tissue engineering. Processes used to form stronger synthetic matrices typically are not suited for cell viability because they are subject to harsh conditions, such as melting or organic solvents. Moreover, synthetic materials typically become much stiffer than those suitable for matching mammalian tissues. The extracellular matrix (ECM) surrounding mammalian cells in tissues is composed of fibers (for example, collagen and elastin) composed of modified proteins that must be produced through synthesis, and they are still in their in vitro lt; / RTI > in vitro ) mechanical properties were not established. In addition, other organisms use protein fibers as a support; The most powerful are the silk threads emitted by the spider. Besides superior strength, the web has very attractive properties such as elasticity and biocompatibility.

Spiders have up to seven different glands that produce various silk types with varying mechanical properties and functions. The dragline silk produced by the major ampullate gland is the toughest fiber and is superior to artificial materials such as tensile steel on a weight basis. The properties of the dragline silk are attractive for the development of new materials for medical or technical purposes such as, for example, scaffolds for cell culture.

Dragline silk is referred to primarily as major ampullate spidroin (MaSp) 1 and 2, but in the case of Araneus diadematus , for example, two major strains referred to as ADF-3 and ADF-4 Lt; / RTI > These proteins have a molecular mass in the range of 200-720 kDa. The gene coding for the dragline protein of Latrodectus hesperus is the only fully characterized gene and the MaSp1 and MaSp2 genes encode 3129 and 3779 amino acids, respectively (Ayoub NA et al . PLoS ONE 2 (6): e514, 2007). The characteristics of dragline silk polypeptides are described in Huemmerich, D. et al . Curr. Biol. 14, 2070-2074 (2004).

Spider drag line silk protein or MaSp is a tripartite composition; A non-repeating N-terminal domain, a central repeating region consisting of many repeated poly-Ala / Gly segments, and a non-repeating C-terminal domain. Generally, it is believed that the repeating region forms an intermolecular contact in silk fibers, although the exact function of the terminal domain is considered to be somewhat obscure. Also, with respect to fiber formation, it is believed that the repeating region is structurally converted from a random coil and a-helical conformation into a beta-sheet structure. The C-terminal region of the speedroin is generally conserved between spider species and silk types. The N-terminal domain of the web is the most conserved region (Rising, A. et al . Biomacromolecules 7, 3120-3124 (2006)).

WO 07/078239 and Stark et al . (Star, M. et al ., Biomacromolecules 8, 1695-1701, (2007)) disclose that a repetitive fragment having a high content of Ala and Gly and a small fragment of a C- ) ≪ / RTI > as well as spider-line proteins. Spider web proteins are spontaneously deformed into a macrostructure of coherent water insoluble, such as a regular polymer such as a fiber, when encountering an interface such as air: water. Small spider-line protein units are sufficient for fiber formation and are essential. Cells from the immortalized cell line are allowed to grow by addition to the pre-formed, visually visible spider silk fiber phase.

Hedhammar, M. et al . (Hedhammar, M. et al ., Biochemistry 47, 3407-3417, (2008)) disclose recombinant N- and C-terminal speedoin domains and four poly-Ala and -Gly- The effect of heat, pH and salt on the structure and coagulation and / or polymerization of the repetitive speed-loop domain containing co-blocks is studied.

WO 2011/129756 discloses methods and cell support materials based on small web-like proteins for eukaryotic cell culture. The protein may contain a variety of short (3 to 5 amino acid residues) cell-binding peptides. A variety of cell types are added onto the pre-formed cell support material.

WO 2012/055854 discloses the preparation of a cell support material comprising a recombinant protein which is a fusion protein between a spider web protein and a longer (more than 30 amino acid residues), non-speed loop polypeptide or protein with desirable binding properties . The cells are added to the pre-formed cell support material and then cultured.

WO 2015/036619 and Widhe, M. et al . (Widhe, M. et al ., Biomaterials 74: 256-266 (2016)) disclose smaller cobweb proteins with useful cell-binding peptides. Again, various cell types are added on the pre-formed cell support material.

Johansson et al. (Johansson et al. , PLOS ONE 10 (6): e0130169 (2015)) disclose the formation of spider web proteins in various physical formats. Pancreatic mouse islets were then placed on top of the web matrix to attach.

Despite these advances in this field, there is still a need for new cell scaffolds in this area. In particular, there is a need for mechanically robust three-dimensional scaffolds for use in culturing integrated eukaryotic cells and tissue engineering in this field.

Summary of the Invention

It is an object of the present invention to provide a cell scaffold with improved cell suitability and flexibility when the cell is about to proliferate.

It is also an object of the present invention to provide a cell support which achieves tissue-like spreading of cultured cells.

It is an object of the present invention to provide a cell support with high cell seeding efficiency which provides fast and viably attached cells.

A further object of the present invention is to provide a cell support with sufficient mechanical strength and suitable stiffness for mammalian tissue engineering.

It is also an object of the present invention to provide a method for providing a cell support under conditions suitable for cell viability.

It is another object of the present invention to provide a cell support in which the cells are integrated throughout the cell support material.

The object of the present invention is also to provide a method which enables co-culture of several cell types in a cell support.

For these and other purposes to be apparent from the following disclosure, the present invention provides a method of culturing eukaryotic cells, comprising the following steps according to a first aspect:

(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;

(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture;

(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells to form a support material for culturing eukaryotic cells; And

(d) maintaining eukaryotic cells within the support material under conditions suitable for cell culture.

In a preferred variation of the method for culturing eukaryotic cells, the silk protein is a web protein.

The present invention provides a creative insight into the ability of the dispersed eukaryotic cells to be incorporated into the silk protein solution prior to assembly into the water insoluble macrostructure of the silk protein so that it can be incorporated throughout the silk-like material during the mild self- . This is in contrast to prior art cell culture methods in which cells are added on pre-formed silk macrostructures.

Advantageously, the formation of macrostructures, including integrated cells, provides high seeding efficiencies that provide fast and viably adherent cells.

Compared to culturing in a hydrogel, the cells become more tissue-like diffusions when incorporated into a silk support using the method according to the invention.

As described herein, it does not matter which specific spider-line protein is used in the present invention. The silk protein is preferably fibroin, such as silk fibroin, or arabic protein.

According to a second aspect, the present invention provides a method for producing eukaryotic cells comprising: (i) a support material for culturing eukaryotic cells; And (ii) a eukaryotic cell that grows integrally with the support material.

(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;

(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture; And

(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells to form a support material for culturing eukaryotic cells.

In a preferred variant of the process for the production of cell culture products, the silk protein is a web of proteins.

According to a third aspect, the present invention provides a water insoluble macroscopic structure of (i) a silk protein that can be assembled into a water insoluble macrostructure, wherein the silk protein selectively encapsulates a cell- ≪ / RTI > And (ii) a eukaryotic cell that grows integrally with the support material.

In a preferred variation of the cell culture product, the silk protein is a web protein.

In a preferred embodiment, the cell culture product can be obtained or obtained by the process according to the invention.

The present invention provides a novel use of a silk protein that can be assembled into a water insoluble macrostructure in the presence of said cells in the formation of a support material for culturing eukaryotic cells according to a fourth aspect; Wherein the support material is a water insoluble macro structure of a silk protein; Wherein the silk protein optionally contains a cell-binding motif.

In a variation of the preferred application, the silk protein is a web protein.

In some preferred embodiments of these and other aspects of the invention, the macrostructure has a shape selected from a fiber, a foam, a film, a fiber mesh, a capsule and a net, preferably a fiber or foam.

In certain preferred embodiments of these and other aspects of the invention, eukaryotic cells are selected from mammalian cells and are preferably selected from the group consisting of primary cells and cell lines such as endothelial cells, fibroblasts, keratinocytes Skeletal muscle satellite cell, skeletal muscle myoblast, smooth muscle cell, umbilical vein endothelial cell, Schwann cell, pancreatic β- Cells, pancreatic islet cells, hepatocytes and glioma-forming cells; And stem cells such as mesenchymal stem cells; Or a combination of at least two different types of mammalian cells.

In certain preferred embodiments of the invention, the silk protein is fibroin, such as silk fibroin.

In some preferred embodiments of the invention, the silk protein is a web protein. In some preferred embodiments of these and other aspects of the invention, the web comprises or consists of protein moieties REP and CT , wherein

REP is a L (AG) _n L, L (AG) _n AL, L (GA) _n L, and L (GA) _n GL 70 to about 300 repetitive fragment of an amino acid residue selected from the group consisting of, where n Is an integer from 2 to 10; Each individual A segment is an amino acid sequence of 8 to 18 amino acid residues wherein 0 to 3 amino acid residues are not Ala and the remaining amino acid residues are Ala; Wherein each individual G segment is an amino acid sequence of 12 to 30 amino acid residues wherein at least 40% of the amino acid residues are Gly; Each separate L segment is a linker amino acid sequence of from 0 to 30 amino acid residues; And CT is a fragment of 70 to 120 amino acid residues having at least 70% identity to SEQ ID NO: 3 or SEQ ID NO: 68; Wherein the selective cell-binding motif is arranged at the end of the spider protein, or between the moieties, or in any moiety, preferably at the end of the spider protein.

In certain preferred embodiments of these and other aspects of the invention, the silk protein is a cell-binding motif such as RGD, IKVAV (SEQ ID NO: 10), YIGSR (SEQ ID NO: 11), EPDIM 13, SEQ ID NO: 14, KYGAASIKVAVSADR, NGEPRGDTYRAY, PQVTRGDVFTM, AVTGRGDSPASS, TGRGDSPA, CTGRGDSPAC, SEQ ID NO: ) And FN _cc (SEQ ID NO: 9); Preferably a cell-binding motif selected from FN _cc , GRKRK, IKVAV, RGD and CTGRGDSPAC, more preferably from FN _cc and CTGRGDSPAC; Wherein FN _cc is C ¹ X ¹ X ² RGDX ³ X ⁴ X ⁵ C ² ; Wherein each X ¹ , X ² , X ³ , X ⁴ and X ⁵ is independently selected from natural amino acid residues other than cysteine; And C ¹ and C ² are connected through a disulfide bond.

Figure 1 shows the sequence alignment of the C-terminal domain of the speedo.
Figure 2 shows a web structure with cell-binding motifs derived from fibronectin.
Figure 3 shows the formation of a silk support containing integrated cells.
Figure 4 shows the metabolic activity of cells in a silk support.
Figure 5 shows cell viability in a silk support.
Figure 6 shows the diffusion of cells in a silk support.
Figure 7 shows the distribution of cells in a silk support.
Figure 8 shows the mechanical properties of silk fibers containing cells.
Figure 9 shows immunofluorescence staining of collagen type I in fibroblasts grown on a silk support.
Figure 10 shows immunofluorescence staining of myotube formation in Hsk cells grown on silk fibers.
Figure 11 shows the presence of various types of cells co-cultured in a silk support.
Figure 12 shows that an islet-like cluster is functional in a silk support.
Figure 13 shows an in vivo image of a silk support containing cells.
Figure 14 shows the distribution of cells in the silk fibers.
15 shows the cell distribution in the silk foam.
Figure 16 shows the growth curves of cells proliferating in silk foam.
Figure 17 shows the staining of living cells integrated in a silk foam.
Figure 18 shows the growth curves of cells proliferating in silk fibers.
Figure 19 shows staining of living cells integrated in silk fibers.
20 shows a growth curve of a cell proliferating in a silk film.
Figure 21 shows images of living cells integrated within the silk film and foam.
Figure 22 shows a micrograph of cells integrated into the silk film and their crystal violet absorption.
Figure 23 shows stem cells differentiated into adipogenic and osteogenic lineages, respectively.
Figure 24 shows the relative gene expression of neuronal progenitor markers in differentiated stem cells.

List of attached sequences

* Widhe M et al., Biomaterials 34 (33): 8223-8234 (2013)

DETAILED DESCRIPTION OF THE INVENTION

The tissue consists of cells integrated into a composite material called the extracellular matrix (ECM). The ECM provides specific support for physical three-dimensional support and cell anchorage. The present inventors have developed a recombinant silk protein functionalized with a motif from the ECM protein fibronectin (FN), which improves the cell-supporting ability of FN-silk formed therefrom. The mild self-assembly process can be used to form various forms of spider web supports, including foams, fibers and films. The mild self-assembly process is surprisingly also useful for forming various forms of fibro-silk, including foams, fibers and films.

Acute damage and trauma with severe tissue loss and impairment lead to problems in the restoration process due to loss of extracellular matrix induction. The healing process is inadequate, and life-support agencies such as the liver can be life-threatening. The liver has a unique ability to self-renew, and can be regenerated if there is time and opportunity. Recombinant cytoplasm can aid in the treatment of hepatic failure by providing a supporting support for the surviving patient's own liver cells. This provides the opportunity to regenerate and repair liver cells and can be a personalized liver graft.

Co-formulations of silk associated with cells from specific tissues (normal or cancer) can also develop a 3D in vitro platform for disease modeling, drug discovery and toxicology. Cancer treatment is targeted at personal medicine due to the complexity of cancer disease. Biomimetic 3D culture of co-formulated cancer and recombinant webs is an example of how it may be possible to screen the progression of cancer and develop a customized method for targeting and destroying cancer-specific therapy-cancer.

The present invention is based on the insight that dispersed mammalian cells can be added to a silk protein solution before being assembled into water-insoluble ordered polymers or macrostructures, thereby integrating into the entire silk-like material . Collection of various mammalian cell types (derived from mice and humans) has been successfully integrated into various silk formats including fibers, foams and films. Silk proteins are fibroin or spider-line proteins. The proliferative capacity of the cells remained in the spider web for more than 2 weeks and there was slight variation when reaching confluence depending on the cell type. Viability was high (> 80%) for all cell types examined, and viability was also confirmed in the innermost part of the material. Observed cell penetration is very beneficial for the formation of artificial tissue structures.

It has been demonstrated herein that the formation of macrostructures with integrated cells, preferably films and foams, provides high seeding efficiencies, resulting in fast and viably adherent cells. Elongated cells with filamentous actin and a defined focal adhesion point show proper attachment within the support. Cryosectioning is used to further confirm the presence of cells in the deepest part of the material. To investigate the mechanical properties, tensile testing of cell-containing gadolinium fibers was performed under physiologically-similar conditions. In vivo images of the cell-containing gill scaffold transplanted into the anterior eye chamber confirmed the maintenance of the cells for 4 weeks in vivo.

Compared to culturing in a hydrogel, the cells exhibit tissue-like spreading as compared to incorporation into a silk support using the method according to the invention.

Most unique tissue types consist of several cell types that are organized together in a complex three-dimensional array with an extracellular matrix surrounding and retaining the cells. Thus, in order to replicate this in an artificial tissue structure, it is important to achieve co-cultivation within the support. It is actually very easy to combine several cell types with the methods described herein for the formation of cell containing silk supports.

According to a first aspect, a method of culturing eukaryotic cells is provided. The method is preferably performed in vitro. The method includes the following steps:

(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;

(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture;

(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells to form a support material for culturing eukaryotic cells; And

(d) maintaining eukaryotic cells within the support material under conditions suitable for cell culture.

Preferably, the eukaryotic cell is a mammalian cell, preferably a human cell comprising primary cells, cell lines and stem cells. Useful examples of primary cells and cell lines include endothelial cells, fibroblasts, keratinocytes, skeletal muscle satellite cells, skeletal muscle myoblasts, smooth muscle cells, umbilical vein endothelial cells, Schwann cells, pancreatic? -Cells, islet cells, hepatocytes and glioma- Forming cells. The stem cells are preferably human pluripotent stem cells (hPSCs) such as embryonic stem cells (ESC) and induced pluripotent cells (iPS). Useful examples of stem cells include mesenchymal stem cells. The cell may also preferably be at least two different mammalian cell types, such as a combination of the above.

In the first step, an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure is provided. Although the composition of the aqueous solution is not critical, it is generally preferred to use a weak aqueous buffer, for example a phosphate buffer having a low or medium ionic strength and a pH in the range of 6-8. The aqueous solution preferably does not contain organic solvents such as hexafluoroisopropanol, DMSO and the like.

In certain preferred embodiments of the invention, the silk protein is fibroin. Fibroin exists in silks produced by spiders, moths such as silkworms and other insects. Preferred fibroin is derived from the genus Bombyx , preferably from the silkworm of Bombyx mori .

In certain preferred embodiments of the invention, the silk protein is a web protein. The term "speed to the (spidroin)" and "spider web protein (spider silk protein)" is in the case of are used interchangeably throughout the specification, typically "MaSp" or Ara Neuss dia Demas tooth (Araneus diadematus) "Quot; ADF ".< / RTI > These large scapular web proteins generally have two types of 1 and 2. These terms also include non-naturally occurring proteins with a high degree of identity and / or similarity to known arabic proteins.

The silk protein optionally contains a cell-binding motif (CBM). The selective cell-binding motif is arranged at the end of the silk protein or in the silk protein, preferably at the N-terminus or the C-terminus of the silk protein.

Once assembled into a macrostructure, the silk protein provides internal solid support activity to the cell. For the avoidance of doubt, the term " macrostructure " refers to a consistent form of silk protein, typically a regular polymer, such as fibers, foams or films, and is not an irregular aggregate or precipitate of the same protein. When the silk protein contains more cell-binding motifs, the resulting macromolecule has both the desired selective cell-binding activity in the cell-binding motif and the internal solid support activity in the silk protein fragment. The binding activity of the silk protein is maintained when the silk protein is structurally rearranged to form a polymeric solid structure. These macrostructures also provide a high and predictable density of cell-binding motifs. For example, the manner in which RGD-functionalized biomaterials stimulate different cellular responses is affected not only by the type of RGD motif used but also by the surface concentration of the resulting ligand. The somewhat smaller silk proteins used in the present study are expected to present a dense surface since each molecule is self-assembled into multilayers with RGD motifs. However, if a less dilute surface concentration is desired, any possible surface density can be achieved simply by mixing the silk proteins with or without the cyclic RGD cell-binding motifs disclosed herein in varying proportions, Lt; / RTI >

The cell-binding motif may comprise, for example, an amino acid sequence selected from the group consisting of RGD, IKVAV (SEQ ID NO: 10), YIGSR (SEQ ID NO: 11), EPDIM (SEQ ID NO: 12) and NKDIL . While RGD, IKVAV and YIGSR are common cell-binding motifs, EPDIM and NKDIL are known as keratinocyte-specific motifs that may be particularly useful in the context of culturing keratinocytes. Other useful cell-binding motifs include GRKRK (SEQ ID NO: 14), KYGAASIKVAVSADR (derived from laminin, SEQ ID NO: 15), NGEPRGDTYRAY (derived from bone sialoprotein, SEQ ID NO: 16) from tropoelastin, PQVTRGDVFTM (Derived from bitronectin, SEQ ID NO: 17), AVTGRGDSPASS (derived from fibronectin, SEQ ID NO: 18), TGRGDSPA (SEQ ID NO: 19) and FN _cc such as CTGRGDSPAC (SEQ ID NO: 20).

Certain related silk structures with cell binding motifs are illustrated in FIG. Figure 2a schematically shows a cytoplasmic protein 4RepCT with different RGD motifs genetically introduced at its N-terminus. 1A shows the RGD-containing peptide (SEQ ID NO: 21) used in Widhe M et al . (Widhe M et al ., Biomaterials 34 (33): 8223-8234 (2013)). &Quot; FN _VS " represents an RGD-containing decapeptide derived from fibronectin (SEQ ID NO: 22). In Figure 1A, " FN _CC " refers to the same peptide in which V and S are exchanged for C (SEQ ID NO: 20). &Quot; FN _SS " refers to the same peptide in which V and S are exchanged to S (SEQ ID NO: 23). Figure IB shows the structure of the ninth and tenth domains of fibronectin, representing a turn loop containing the RGD motif. Figure 1c shows a structural model of an RGD loop taken from fibronectin, in which residues V and S were mutated to C (modified from 1FNF.pdb).

In the most general form, FN _cc is C ¹ X ¹ X ² RGDX ³ X ⁴ X ⁵ C ² (SEQ ID NO: 9), wherein each of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is independently cysteine Other natural amino acid residues; C ¹ and C ² are connected through a disulfide bond. FN _cc is a modified cell-binding motif that forms a disulfide bridging to mimic the [alpha] 5 [beta] l -specific RGD loop motif of fibronectin by positioning the cysteine at the correct position adjacent to the RGD sequence to create a similar type of turn loop. These cyclic RGD cell-binding motifs increase the cell adhesion efficiency to a matrix containing a cell-binding motif, for example, a web made of recombinant protein. The term " cyclic " as used herein refers to peptides in which two amino acid residues are covalently bound through their side chains, more particularly through a disulfide bond between the two cysteine residues. The cyclic RGD cell-binding motif FN _cc promotes both proliferation of primary cells and migration by primary cells. Human primary cells cultured on cell support material containing cyclic RGD cell-binding motifs exhibit increased adhesion, diffusion, stress fiber formation and local attachment compared to the same material containing linear RGD peptides.

In a preferred embodiment of FN _cc , each of X ¹ , X ² , X ³ , X ⁴ and X ⁵ is independently selected from the group consisting of amino acids comprising G, A, V, S, T, D, E, M, P, Lt; / RTI > In another preferred embodiment of FN _cc , each of X ¹ and X ³ is independently selected from the group of amino acid residues consisting of G, S, T, M, N and Q; Each of X ² , X ⁴ and X ⁵ is independently selected from the group of amino acid residues consisting of G, A, V, S, T, P, N and Q. In certain preferred embodiments of FN _cc , X ¹ is selected from the group of amino acid residues consisting of G, S, T, N and Q; X ³ is selected from the group of amino acid residues consisting of S, T and Q; Each of X ² , X ⁴ and X ⁵ is independently selected from the group of amino acid residues consisting of G, A, V, S, T, P and N. In some preferred embodiments of FN _cc , X ¹ is S or T; X ² is G, A or V; Preferably G or A; More preferably G; X ³ is S or T; Preferably S; X ⁴ is G, A, V or P; Preferably G or P; More preferably P; X < ⁵ > is G, A or V; Preferably G or A; More preferably, it is A.

In certain preferred embodiments of the FN _cc , the cell-binding motif comprises the amino acid sequence CTGRGDSPAC (SEQ ID NO: 20). A further preferred cyclic RGD cell-binding motif according to the present invention exhibits at least 60%, such as at least 70%, such as at least 80%, such as at least 90% identity to CTGRGDSPAC (SEQ ID NO: 20) 10 is always C; Position 4 is always R; Position 5 is always G; Position 6 is always D; Positions 2-3 and 7-9 are never cysteine. The positions that are not the same among positions 2-3 and 7-9 can be freely selected as shown above.

Preferred groups of cell-binding motifs are FN _cc , GRKRK, IKVAV, and RGD, particularly FN _cc , such as CTGRGDSPAC.

The spiderweb protein preferably comprises or consists of protein moieties REP and CT . The preferred arachnoid protein has the structure REP-CT . Another preferred spider-line protein has the structure REP-CT . Selective cell-binding motifs are arranged at the end of the cobweb protein, or between the moieties, or in any moiety, preferably at the N-terminus or C-terminus of the cobra protein.

REP is L (AG) _n L, L (AG) _n AL, and L (GA) _n L and L (GA) _n GL, 70 to about 300 repetitive fragment of an amino acid residue selected from the group consisting of, where

n is an integer from 2 to 10;

Each individual A segment is an amino acid sequence of 8 to 18 amino acid residues wherein 0 to 3 of the amino acid residues are not Ala and the remaining amino acid residue is Ala;

Wherein each individual G segment is an amino acid sequence of 12 to 30 amino acid residues wherein at least 40% of the amino acid residues are Gly; And

Each separate L segment is a linker amino acid sequence of from 0 to 30 amino acid residues; And

CT is a fragment of 70 to 120 amino acid residues having at least 70% identity to SEQ ID NO: 3 or SEQ ID NO: 68.

The silk protein according to the present invention is preferably a recombinant protein, i.e., a recombinant nucleic acid, i.e., a protein made by expression from DNA or RNA that is artificially produced (genetic engineering) by binding two or more nucleic acid sequences that will not normally coexist to be. The web proteins according to the present invention are preferably recombinant proteins, so they are not the same as naturally occurring proteins. In particular, they are preferably not the cobweb proteins according to the invention, as the wild type speedloins are not expressed from the recombinant nucleic acid as indicated above. The combined nucleic acid sequences encode different proteins, partial proteins or polypeptides with specific functional properties. The resulting recombinant protein is a single protein with functional properties derived from each of the original protein, partial protein or polypeptide.

Spiderweb proteins typically comprise 140 to 2000 amino acid residues, such as 140 to 1000 amino acid residues, such as 140 to 600 amino acid residues, preferably 140 to 500 amino acid residues, such as 140 to 400 amino acid residues. Smaller sizes are advantageous because longer proteins containing spider web protein fragments form amorphous aggregates requiring the use of strong (harsh) solvents for solubilization and polymerisation.

The web may contain one or more linker peptides, or L segments. The linker peptide (s) may be attached to the surface of the cell membrane between any of the motifs of the web protein, for example between the REP and CT moieties, or at the terminal end of the spider protein, Lt; / RTI > The linker (s) may provide a spacer between the functional units of the web protein, but may also constitute a handle, for example a His and / or Trx tag, for identification and purification of the web protein . If the spiderweb protein contains two or more linker peptides for the identification and purification of the spider-line proteins, they are preferably separated by a spacer sequence, for example His ₆ -space-His ₆ -. Linkers can also constitute signal peptides, such as signal recognition particles, which send and / or send spider proteins to the membrane or secrete spider proteins from the host cells into the surrounding media. The web may also contain cleavage sites in its amino acid sequence, which allows cleavage and removal of the linker (s) and / or other related moieties. Cleavage sites for chemical agents such as hydroxylamine between CNBr and Asn-Gly residues after various cleavage sites such as Met residues, cleavage sites for proteolytic enzymes such as thrombin or protease 3C, Self-splicing sequences such as intein self-splicing sequences are known to those skilled in the art.

The fastroin fragment and the cell-binding motif are linked directly or indirectly to each other. Direct linking means direct covalent linkage between moieties without an intervening sequence such as a linker. An indirect link also means that the moiety is linked by a covalent bond but there is an intervening sequence such as a linker and / or one or more additional moieties, e. G. 1-2 NT moieties.

The cell-binding motif may be arranged internally or at an end, either at the C-terminus or at the N-terminus, at either end of the web protein. It is preferred that the cell-binding motif is arranged at the end of the N-terminus of the spider web protein. If the spider web protein contains one or more linker peptide (s), e.g. His or Trx tag (s), for identification and purification of the spider web protein, it is preferred to be arranged at the N-terminal end of the spider web protein.

Preferred arachidic proteins have the form of N-terminal aligned cell-binding motifs linked to REP moieties by 0-30 amino acid residues, such as linker peptides of 0-10 amino acid residues. Alternatively, the web has an N-terminal or C-terminal linker peptide, which may contain a purification tag such as a His tag and a cleavage site.

The protein moiety REP is a fragment with repetitive characters, alternating between alanine-rich and glycine-rich segments. The REP fragment generally contains more than 70, such as greater than 140 and less than 300, preferably less than 240, such as less than 200 amino acid residues, and may contain several L (linker) segments, A (alanine-rich) segments and G (glycine-rich) segments. Typically, the optional linker segment is located at the end of the REP fragment, while the remaining segments are in turn rich in alanine and rich in glycine. Thus, REP fragments can generally include any of the following structures, where n is an integer: < RTI ID = 0.0 >

L ( AG ) _n L , such as LA ₁ G ₁ A ₂ G ₂ A ₃ G ₃ A ₄ G ₄ A ₅ G ₅ L ;

L ( AG ) _n AL , such as LA ₁ G ₁ A ₂ G ₂ A ₃ G ₃ A ₄ G ₄ A ₅ G ₅ A ₆ L ;

L ( GA ) _n L , such as LG ₁ A ₁ G ₂ A ₂ G ₃ A ₃ G ₄ A ₄ G ₅ A ₅ L ; or

L ( GA ) _n GL , such as LG ₁ A ₁ G ₂ A ₂ G ₃ A ₃ G ₄ A ₄ G ₅ A ₅ G ₆ L.

Whether the alanine-rich or glycine-rich segment is adjacent to the N-terminal or C-terminal linker segment is not important. n is an integer of 2 to 10, preferably 2 to 8, further preferably 4 to 8, more preferably 4 to 6, that is, n = 4, n = 5 or n =

In some embodiments, the alanine content of the REP fragment is greater than 20%, preferably greater than 25%, more preferably greater than 30% and less than 50%, preferably less than 40%, and more preferably less than 35%. It is believed that the higher alanine content provides fibers that are stiffer and / or stronger and / or less extended.

In certain embodiments, the REP fragment is free of proline residues. That is, there is no Pro residue in the REP fragment.

Turning now to segment constituting the REP fragments, each segment is stressed that the individual and, that specific REP any two A segments of the fragments, and any two G segments, or any two L segment of the may not be the same as or identical. Thus, it is not a common feature of speed loops that each type of segment is the same within a particular REP fragment. Rather, the disclosure below provides guidance to those of ordinary skill in the art how to design individual segments and to collect them into REP fragments that are part of the functional spider web protein useful for cell support materials.

Each individual A segment is an amino acid sequence comprising 8 to 18 amino acid residues. It is preferred that each individual A segment contain 13 to 15 amino acid residues. Also, many or more than two A segments contain 13 to 15 amino acid residues, and a few A segments such as one or two fragments comprise 8 to 18, such as 8 to 12 or 16 to 18 amino acid residues . Most of these amino acid residues are alanine residues. More specifically, 0 to 3 amino acid residues are not alanine residues, and the remaining amino acid residues are alanine residues. Thus, all amino acid residues within each individual A segment are alanine residues, with the exception of 1, 2 or 3 amino acid residues which may or may not be any amino acids. It is preferred that the alanine-substituted amino acid (s) are natural amino acids, preferably selected from the group of serine, glutamic acid, cysteine and glycine, more preferably serine, wherein one or more of the A segments are all alanine segments , And the remaining A segment can contain 1-3 non-alanine residues such as serine, glutamic acid, cysteine or glycine.

In one embodiment, each A segment contains 13-15 amino acid residues comprising 10-15 alanine residues and 0-3 non-alanine residues as described above. In a more preferred embodiment, each A segment contains 13-15 amino acid residues comprising 12-15 alanine residues and 0-1 non-alanine residues as described above.

Each individual A segment has amino acid residues 7-19, 43-56, 71-83, 107-120, 135-147, 171-183, 198-211, 235-248, 266-279, 294-306 , 330-342, 357-370, 394-406, 421-434, 458-470, 489-502, 517-529, 553-566, 581-594, 618-630, 648-661, 676-688, 712 -725, 740-752, 776-789, 804-816, 840-853, 868-880, 904-917, 932-945, 969-981, 999-1013, 1028-1042 and 1060-1073 It is preferred to have at least 80%, preferably at least 90%, more preferably 95%, most preferably 100% identity to the amino acid sequence. Each sequence of this group corresponds to a segment of the naturally occurring sequence of the Euprosthenops australis MaSp1 protein derived from the cloning of the corresponding cDNA, see WO2007 / 078239. Alternatively, each individual A segment is at least 80%, preferably at least 80%, preferably at least 80%, at least 80%, at least 80%, at least 80% 90%, more preferably 95%, most preferably 100% identity. Each sequence in this group corresponds to an expressed, non-natural segment of the gabbro protein, and the protein is capable of forming silk fibers under appropriate conditions. Thus, in certain embodiments of the speedologens, each individual A segment is identical to the amino acid sequence selected from the above-mentioned amino acid segment. Without being bound to any particular theory, it is expected that the A segment according to the present invention will form a helical structure or a beta sheet.

Further, from the experimental data, it was concluded that each individual G segment is an amino acid sequence of 12 to 30 amino acid residues. Preferably, each individual G segment comprises 14 to 23 amino acid residues. At least 40% of the amino acid residues of each G segment are glycine residues. Typically, the glycine content of each individual G segment is in the range of 40-60%.

Each individual G- segment comprises amino acid residues 20-42, 57-70, 84-106, 121-134, 148-170, 184-197, 212-234, 249-265, 280-293, 307-329 of SEQ ID NO: , 343-356, 371-393, 407-420, 435-457, 471-488, 503-516, 530-552, 567-580, 595-617, 631-647, 662-675, 689-711, 726 -739, 753-775, 790-803, 817-839, 854-867, 881-903, 918-931, 946-968, 982-998, 1014-1027, 1043-1059 and 1074-1092 It is preferred to have at least 80%, preferably at least 90%, more preferably 95%, most preferably 100% identity to the amino acid sequence. Each sequence of this group corresponds to a segment of the naturally occurring sequence of the Euprosthenops australis MaSp1 protein derived from the cloning of the corresponding cDNA, see WO2007 / 078239. Alternatively, each individual G- segment is at least 80%, preferably at least 80%, preferably at least 80%, at least 80%, at least 80%, at least 80% 90%, more preferably 95%, most preferably 100% identity. Each sequence in this group corresponds to an expressed, non-natural segment of the gabbro protein, and the protein is capable of forming silk fibers under appropriate conditions. Thus, in certain embodiments of the speedologens in the cell support material, each individual G segment is identical to the amino acid sequence selected from the above-mentioned amino acid segments.

In certain embodiments, the first two amino acid residues of each G segment are not -Gln-Gln-.

The G segment has three subtypes. This classification is based on Is based on careful analysis of the osthenops australis MaSp1 protein sequence (see WO 2007/078239), and the information has been used and verified in the production of novel non-natural spider-line proteins.

The first subtype of the G segment is represented by the amino acid single-letter common sequence GQG (G / S) QGG (Q / Y) GG (L / Q) GQGGYGQGA GSS (SEQ ID NO: 6). This first and generally longest G segment subtype typically contains 23 amino acid residues, but may contain at least 17 amino acid residues and may lack a charged residue or contain a single charged residue . Thus, it is contemplated that this first G segment subtype may contain 17-23 amino acid residues, but may contain only 12 or even 30 amino acid residues. Without being bound by any particular theory, it is expected that this subtype will form a coiled structure or a 3 ₁ -helical structure. Representative G segments of this first subtype are the amino acid residues 20-42, 84-106, 148-170, 212-234, 307-329, 371-393, 435-457, 530-552, 595-617 , 689-711, 753-775, 817-839, 881-903, 946-968, 1043-1059, and 1074-1092. In certain embodiments, the first two amino acid residues of each G segment of this first subtype according to the invention are not -Gln-Gln-.

The second subclass of the G segment is represented by the amino acid single-letter common sequence GQGGQGQG (G / R) Y GQG (A / S) G (S / G) S (SEQ ID NO: 7). This second and generally mid-sized G- segment subtype typically contains 17 amino acid residues and either lacks charged residues or contains a single charged residue. This second G segment subtype is thought to contain only 14 or 20 amino acid residues, but only 12 or as many as 30 amino acid residues. Without being bound to any particular theory, this subtype is expected to form a coil structure. Representative G segments of this second subtype are amino acid residues 249-265, 471-488, 631-647 and 982-998 of SEQ ID NO: 5.

The third subtype of the G segment is represented by the amino acid single-letter common sequence G (R / Q) GQG (G / R) YGQG (A / S / V) GGN (SEQ ID NO: 8). This third G segment subtype typically contains 14 amino acid residues and is generally the shortest of the G segmented subtypes. This subtype of the third G segment is thought to contain 12 to 17 amino acid residues, but may contain as many as 23 amino acid residues. Without being bound by any particular theory, this subtype is expected to form a turn structure. Representative G segments of this third subtype are the amino acid residues 57-70, 121-134, 184-197, 280-293, 343-356, 407-420, 503-516, 567-580, 662-675 , 726-739, 790-803, 854-867, 918-931, 1014-1027.

Thus, in a preferred embodiment of the speedoin in the cell support material, each individual G segment is at least 80%, preferably 90%, more preferably at least 80%, more preferably at least 80% Has 95% identity.

In one embodiment of the alternating sequence of the A and G fragments of the REP fragment, all the second G segments are the first subtype whereas the remaining G segments are the third subtype, for example ... A ₁ G _short A ₂ G _long A ₃ G _short A ₄ G _long A ₅ G _short .... In another embodiment of the REP fragment, both a segment G of the second subtype is inserted through, for example, A ₁ ... A _short G ₂ G ₃ G _{mid long} A A G ₄ G ₅ A _{short long} .. It interferes with the regularity of the G - segment as.

Each separate L segment represents an optional linker amino acid sequence that can contain from 0 to 30 amino acid residues, such as from 0 to 20 amino acid residues. This segment is optional and is not critical to the function of the web protein, but its presence still allows fully functioning web proteins and their polymers to form fibers, films, foams and other structures. There is also a linker amino acid sequence present in the repetitive portion (SEQ ID NO: 5) of the deduced amino acid sequence of the MaSp1 protein from Euprosthenops australis . In particular, the amino acid sequence of the linker segment may be similar to any of the A or G segments described, but is generally not sufficient to meet the criteria as defined herein.

As shown in WO 2007/078239, linker segments arranged at the C-terminal portion of the REP fragment can be represented by the alanine-rich amino acid single-letter sequence ASASAAASAA STVANSVS (SEQ ID NO: 32) and ASAASAAA (SEQ ID NO: 33). In fact, the second sequence can be regarded as an A segment according to the definition of the present invention, whereas the first sequence has a high similarity to the A segment according to this definition. Another example of a linker segment is glycine-rich and has the one-letter amino acid sequence GSAMGQGS (SEQ ID NO: 34), which has a high similarity to the G segment according to the definition herein. Another example of a linker segment is SASAG (SEQ ID NO: 35).

Representative L segments include amino acid residues 1-6 and 1093-1110 of SEQ ID NO: 5; And amino acid residues 159-165 of SEQ ID NO: 2, but those of ordinary skill in the art will readily recognize that there are many suitable alternative amino acid sequences for these segments. In one embodiment of the REP fragment, one of the L fragments contains zero amino acids, i.e. one of the L fragments is void. In another embodiment of the REP fragment, both L segments contain zero amino acids, i.e. both L segments are empty. Thus, these embodiments of the REP fragment in accordance with the present invention can be represented schematically as _{follows: (AG) n L, (} AG) n AL, (GA) n L, (GA) n GL; _{L (AG) n, L (} AG) n A, L (GA) n, L (GA) n G; And _{(AG) n, (AG)} n A, (GA) n, (GA) n G. Any of these REP fragments is suitable for use with any CT fragment as defined below.

The CT fragment of the speedoin in the cell support material has a high similarity to the C-terminal amino acid sequence of the web protein. As shown in WO2007 / 078239, this amino acid sequence is well conserved among a variety of species and spider proteins, including MaSp1, MaSp2 and MiSp (small bedpeptide). The common sequence of the C-terminal region of MaSp1 and MaSp2 is provided as SEQ ID NO: In Figure 1, the MaSp proteins (SEQ ID NOS: 36-66) presented in Table 1 are sorted and marked according to the applicable GenBank access light control entry.

Speedlowin  CT fragments Species and speedroin Control (Entry) Euprosthenops sp MaSp1 (Pouchkina-Stantcheva *) Cthyb_Esp Euprosthenops australis MaSp1 (SEQ ID NO: 3) CTnat_Eau Argiope trifasciata MaSp1 AF350266_At1 Cyrtophora moluccensis Sp1 AY666062_Cm1 Latrodectus geometricus MaSp1 AF350273_Lg1 Latrodectus hesperus MaSp1 AY953074_Lh1 Macrothele holsti Sp1 AY666068_Mh1 Nephila clavipes MaSp1 U20329_Nc1 Nephila pilipes MaSp1 AY666076_Np1 Nephila madagascariensis MaSp1 AF350277_Nm1 Nephila senegalensis MaSp1 AF350279_Ns1 Octonoba varians Sp1 AY666057_Ov1 Psechrus sinensis Sp1 AY666064_Ps1 Tetragnatha kauaiensis MaSp1 AF350285_Tk1 Tetragnatha versicolor MaSp1 AF350286_Tv1 Araneus bicentenarius Sp2 ABU20328_Ab2 Argiope amoena MaSp2 AY365016_Aam2 Argiope aurantia MaSp2 AF350263_Aau2 Argiope trifasciata MaSp2 AF350267_At2 Gasteracantha mammosa MaSp2 AF350272_Gm2 Latrodectus geometricus MaSp2 AF350275_Lg2 Latrodectus hesperus MaSp2 AY953075_Lh2 Nephila clavipes MaSp2 AY654293_Nc2 Nephila madagascariensis MaSp2 AF350278_Nm2 Nephila senegalensis MaSp2 AF350280_Ns2 Dolomedes tenebrosus Fb1 AF350269_DtFb1 Dolomedes tenebrosus Fb2 AF350270_DtFb2 Araneus diadematus ADF-1 U47853_ADF1 Araneus diadematus ADF-2 U47854_ADF2 Araneus diadematus ADF-3 U47855_ADF3 Araneus diadematus ADF-4 U47856_ADF4

* Comparative Biochemistry and Physiology, Part B 138: 371 -376 (2004)

It is not critical whether a particular CT fragment is present in the web protein in the cell support material. Thus, the CT fragment may be an amino acid sequence as shown in Figure 1 and Table 1 or any of the sequences with high similarity, such as the MiSp CT fragment sequence number from Araneus ventricosus (Genbank et al. Control AFV 31615) 68 < / RTI > A wide variety of C-terminal sequences can be used for the spider-line proteins.

The sequence of the CT fragment is at least 50% identical, preferably at least 60%, more preferably at least 65% identical, or even at least 70% identical to SEQ ID NO: 4, which is a common amino acid sequence based on the amino acid sequence of FIG. Have the same identity.

A representative CT fragment is the Euprosthenops australis sequence of SEQ ID NO: 3 or the amino acid residue 180-277 of SEQ ID NO: 27. Another exemplary CT fragment is SEQ ID NO: 68, which is the MiSp sequence. Thus, in one embodiment, the CT fragment comprises at least 70%, such as at least 80%, at least 70%, at least 70%, at least 70% %, Such as at least 85%, preferably at least 90%, such as at least 95%. For example, the CT fragment may be SEQ ID NO: 3, amino acid residues 180-277 of SEQ ID NO: 27, or any individual amino acid sequence of FIG. 1 and Table 1, or SEQ ID NO: 68.

CT fragments typically comprise 70 to 120 amino acid residues. The CT fragment preferably contains at least 70, or more than 80, preferably more than 90 amino acid residues. It is also preferred that the CT fragment contains a maximum of 120 or less than 110 amino acid residues. A typical CT fragment contains about 100 amino acid residues.

The term "% identity " as used herein is calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al , Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made to the window that corresponds to the shortest of the sorted sequences. The amino acid residues at each position are compared and the percentage of positions in the query sequence that have the same correspondence in the target sequence is reported as% identity.

The term "% similarity ", as used herein, refers to the hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met and Cys being similar; The basic residues Lys, Arg and His are similar; The acidic residues Glu and Asp are similar; Is calculated as described above for "% identity ", except that residues Gln, Asn, Ser, Thr and Tyr which are not hydrophilic are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

Throughout this specification, alternative embodiments according to the present invention fulfill the corresponding percentages of similarity instead of the specified percentages of identity. Other alternative embodiments satisfy a specified percentage of identity selected from the group of preferred percentages of identity for each sequence as well as a percentage of another high similarity. For example, the sequence may be 70% similar to another sequence; May be 70% identical to another sequence; It may be 70% identical and 90% similar to another sequence.

In a preferred arachnoid protein according to the present invention, the REP-CT fragment is at least 70%, such as at least 80%, such as at least 80%, more preferably at least 80% 85%, preferably at least 90%, such as at least 95%.

In one preferred arachnoid protein according to the present invention, the protein has at least 70%, such as at least 80%, such as at least 85%, preferably at least 90%, such as at least 95% identity to SEQ ID NO: 25, 27 or 69 . In a particularly preferred embodiment, the spider web protein according to the invention is SEQ ID NO: 25, 27 or 69.

The cell support material according to the invention preferably comprises a protein or peptide according to the invention which exhibits a cyclic RGD cell-binding motif. The cyclic RGD cell-binding motif may be exposed from short synthetic peptides or long synthetic or recombinant proteins, which may be attached or attached to a matrix or support.

The cell support material preferably comprises a protein polymer and the protein polymer comprises a silk protein according to the invention as a repeating structural unit, i.e. the protein polymer comprises or consists of a polymer of the silk protein according to the invention. This means that the protein polymer comprises or consists of a plurality of ordered silk proteins according to the invention, typically more than 100 silk protein units, for example, more than 1000 silk protein units. In a preferred embodiment, the cell support material according to the invention consists of a protein polymer.

The magnitude of the silk protein unit of the polymer means that the protein polymer obtains a significant size. In a preferred embodiment, the protein polymer has a size of at least 0.01 [mu] m at least in two dimensions. Thus, the term " protein polymer " as used herein refers to a silk protein polymer having a thickness of at least 0.01 [mu] m, such as at least 0.1 [mu] m, To macroscopic polymers having a thickness of up to 10 [mu] m. The term " protein polymer " does not include unstructured aggregates or precipitates. It is understood that the protein polymers according to the present invention are not soluble in solid form, i.e. water, while the monomers / dimers of the web are water soluble. The protein polymer comprises the monomers of the silk proteins according to the invention as repeating structural units.

The protein polymers according to the invention are typically provided in a physical form selected from the group consisting of fibers, films, coatings, foams, webs, fibers-meshes, spheres and capsules. According to one embodiment, the protein polymer according to the invention is preferably a fiber, film, or fiber-mesh. According to certain embodiments, the protein polymer preferably has a three-dimensional morphology, such as a foam or a fiber-mesh. One preferred embodiment comprises a thin (typically 0.01 - 0.1 탆 thick) coating made of a protein polymer useful for coating stents and other medical devices. The term " foam " includes porous foams having channels connecting the bubbles of the foam, sometimes even to the extent that they can be regarded as a three-dimensional network or mesh of fibers do.

In a preferred embodiment, the protein polymer is a physical form of a self-supporting matrix, such as a free-standing film. This is very useful, for example, if necessary, because it allows delivery of the cell sheet in vivo, for example, as a cell sheet that needs to be delivered to the wound site, for example.

The fiber, film or fiber-mesh typically has a thickness of at least 0.1 μm, preferably at least 1 μm. The fiber, film or fiber-mesh preferably has a thickness in the range of 1 - 400 μm, preferably 60 - 120 μm. The fibers preferably have a length in the range of 0.5 to 300 cm, preferably 1 to 100 cm. Other preferred ranges are 0.5 - 30 cm and 1 - 20 cm. Fibers have the ability to remain intact during physical manipulation and can be used for spinning, weaving, twisting, crocheting and the like procedures. The film is advantageous in that it is consistent and adheres to plastics in solid structures, e.g., microtiter plates. The properties of this film facilitate washing and regeneration procedures and are very useful for separation purposes.

The spider web proteins according to the present invention include internal solid support activities in REP-CT moieties and optionally also cell-binding activities in cell-binding motifs, which are used in cell support materials. The cell support material provides a high and predictable density of selective interacting activity for organic targets. Since all expressed protein moieties are associated with the cell support material, loss of valuable protein moieties with selective interacting activity is minimized.

Polymers formed from the silk proteins according to the present invention are solid structures and are useful for their useful combination of physical properties, especially high strength, elasticity and low weight. Particularly useful features are that the REP-CT moiety of the spider web protein is biochemically robust and suitable for regeneration with, for example, acids, bases or chaotropic agents, and heat sterilization, e.g., 120 Lt; 0 > C for 20 minutes. Polymers are also useful for their ability to support cell adhesion and growth.

The properties derived from the REP-CT moiety are attractive for the development of new materials for medical or technical purposes. In particular, the cell support material according to the present invention is useful as a support for cell immobilization, cell culture, cell differentiation, tissue engineering and induced cell regeneration. They are also useful in preparative and analytical separation procedures such as chromatography, cell capture, selection and culture, active filters and diagnostics. The cell support material according to the present invention is also useful as a coating, for example, in medical devices such as implants and stents.

In a preferred embodiment, the cell support material comprises a protein polymer, which consists of a silk protein according to the invention as a repeating structural unit. And, in a further preferred embodiment, the cell support material is a protein polymer, which consists of a silk protein according to the invention as a repeating structural unit. Silk proteins are fibroin or spider-line proteins.

In the second step, an aqueous mixture of eukaryotic sample and silk protein is prepared. This is preferably achieved by mixing the aqueous solution from the previous step with a liquid cell suspension, or by dispersing the cell pellets. The liquid component of the aqueous mixture should be suitable for each eukaryotic cell in terms of buffering capacity, ionic strength and pH. Media suitable for cell culture and cell handling are well known in the art and are, for example, DMEM, hams nutrient mixture, Minimal Essential Medium Eagle, and RPMI.

Preferably, the eukaryotic cell is a mammalian cell, preferably a primary cell, a cell line, and a human cell comprising stem cells. Useful examples of primary cells and cell lines include endothelial cells, fibroblasts, keratinocytes, skeletal muscle satellite cells, skeletal muscle myoblasts, smooth muscle cells, umbilical vein endothelial cells, Schwann cells, pancreatic beta cells, islet cells, hepatocytes and glioma-forming Cells. The stem cells are preferably human pluripotent stem cells (hPSCs) such as embryonic stem cells (ESC) and induced pluripotent stem cells (iPS). Useful examples of stem cells include mesenchymal stem cells. The cell may also preferably be at least two different mammalian cell types, such as a combination of the above.

In the second step, it is important that the silk protein remains dissolved in the aqueous mixture. The term " dissolved " means that a cell is added to a silk protein before the silk assembly process develops, when the silk protein primarily forms a bond with surrounding water molecules. As the silk assembly process evolves, there is irreversible formation of regular polymers with mainly intramolecular and intermolecular bonds between the silk proteins. Polymerization is a continuous process, but according to the present invention, it is believed that considering the desired final format of the final macrostructure, the cells should be added to the dissolved silk protein as soon as possible. It is preferred that the cells are added when at least a portion, preferably most or even substantially all of the silk protein remains dissolved. Thus, for example, if the desired format is foam, the cell should be added to the wet foam before bubbling occurs, or when the foam is freshly formed by introducing air into the liquid, rather than when the foam is polymerized into the silk macrostructure.

Alternatively, the aqueous mixture may contain additional ingredients that are preferably incorporated into the macrostructure. For example, the aqueous mixture may contain cell-binding proteins and polypeptides, such as laminin.

In the third step, the silk protein can be assembled into a water-insoluble macrostructure in the presence of eukaryotic cells. The protein construct according to the invention is spontaneously assembled from the silk protein according to the invention under suitable conditions and the assembly into the polymer is carried out under shear and / or between two different phases, for example between solid and liquid phases, , Or by the presence of a hydrophobic / hydrophilic interface, for example a mineral oil-water interface. The presence of the generated interface stimulates the polymerization reaction in the region surrounding the interface or interface, and the region extends into the liquid medium to initiate polymerization at the interface or at the interface region. Various protein structures can be produced by adjusting conditions during assembly. For example, if assembly occurs in a gently shaking container from side to side, the fibers are formed at the air-water interface. If the mixture is left to stand, the film is formed at the air-water interface. If the mixture is evaporated, the film is formed on the bottom of the container. If oil is added to the top of the aqueous mixture, the film is formed at the oil-water interface if left to stand or shake. If the mixture is foamed, for example by bubbling or whipping air, the foam is stable and solidifies over time. The new macrostructure can be formed in any suitable cell culture well. Optionally, the culture well surface is pre-coated with a silk macrostructure or other material, such as gelatin.

Assembly into water insoluble macrostructures results in the formation of support materials for culturing eukaryotic cells. Thus, the exact cells to be cultured are already present during assembly of the support material and are incorporated into the cellular material. As a result, the cells are embedded in a spider web macro structure. This has a beneficial effect in terms of viability, proliferative capacity, cell diffusion and attachment in subsequent cell cultures. In addition, the coexistence of cells in the assembly of macrostructures achieves the formation of cavities and pores in support materials that would otherwise not be present.

In a fourth step, the eukaryotic cells are maintained in the support material under conditions suitable for cell culture, well known to those skilled in the art and exemplified herein. This advantageously allows the cells to grow integrated with the support material. This means that the cells grow not only on the surface of the support material, but also within cavities and voids in the support material formed by the co-existence of air bubbles and cells in the assembly of the macro structure.

According to a second aspect, the present invention relates to a method for the production of (i) a support material for culturing eukaryotic cells; And (ii) a eukaryotic cell that grows integrally with the support material. The method is preferably carried out in vitro . The method includes the following steps:

(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;

(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture; And

(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells to form a support material for culturing eukaryotic cells.

Preferred embodiments and variations of the manufacturing method are evident from the above disclosure of the method of culturing eukaryotic cells comprising corresponding steps.

According to a third aspect, the present invention provides a water insoluble macrostructure of (i) a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein is selected from the group consisting of eukaryotic cells A support material for culturing cells; And (ii) a eukaryotic cell that grows integrally with the support material.

This means that the cells adhere not only to the surface of the support material, but also to cavities and pores in the support material formed by coexistence of cells, for example in the assembly of macroscopic structures.

Preferred embodiments and modifications of the cell culture products are evident from the above disclosure of methods of culturing eukaryotic cells comprising corresponding features.

In a preferred embodiment, the cell culture product according to the invention can be obtained or obtained by the process according to the invention. The coexistence of cells in the assembly of macrostructures achieves the formation of voids and voids in the support material that would otherwise not be present.

According to a fourth and final aspect, the present invention provides a novel use of a silk protein that can be assembled into a water-insoluble macrostructure in the formation of a support material for culturing eukaryotic cells in the presence of cells, Is a water insoluble macro structure of silk protein; Wherein the silk protein optionally contains a cell-binding motif. The use is preferably carried out in vitro.

Preferred embodiments and variations of uses are evident from the above disclosure of methods of culturing eukaryotic cells comprising corresponding features.

In summary, a novel method for the formation of cell-containing silk supports has been developed wherein the cells are added to the silk protein before the silk assembly process is developed. The following examples show how cells are affected by integration into a silk support in terms of viability, proliferative capacity, cell diffusion and attachment. To investigate the generality of the method, a broad repertoire of mammalian cells (Table 2) ranging from stable cell lines to primary cells in both mouse and human origin was analyzed. The maintenance of specific cell functions of certain cell types, such as the production, differentiation, and response to glucose stimulation of extracellular matrix components was also confirmed.

Tested mammalian cells silk
form Motif Cell type ^* Cell viability ^** multiplication ^*** Cell integration Textiles: Air FN _CC HSkMC +++ +++ has exist,
Frozen section
And confocal Textiles: Oils FN _CC HSkMC +++ +++ Yes, frozen section Textiles: Air FN _CC HSkMC:
HDMEC +++ ++ Yes, frozen section Textiles: Air FN _CC HDMEC +++ +++ Yes, frozen section Textiles: Oils GRKRK HSkMC + ++ Yes, frozen section Textiles: Oils GRKRK HSkMC:
HDMEC + ++ Yes, frozen section Textiles: Air FN _CC HSMM +++ ++ Yes, frozen section Textiles: Air FN _CC HSMM:
HDMEC +++ ++ Yes, frozen section Textiles: Air FN _CC HDFn:
HDMEC +++ +++ Yes, frozen section Textiles: Air FN _CC HDFn +++ Yes, frozen section Textiles: Oils FN _CC HDFn Yes, frozen section Foam FN _CC HDFn ++ Textiles: Oils FN _CC HaCaT Yes, frozen section Textiles: Air FN _CC HaCaT Yes, frozen section Foam FN _CC HaCaT +++ ++ Foam FN _CC mEC +++ ++ Yes, frozen section Textiles: Air FN _CC mEC +++ ++ Foam FN _CC mMSC +++ ++ Yes, frozen section,
Confocal Textiles: Air FN _CC mMSC +++ ++ Yes, frozen section Textiles: Oils FN _CC mMSC ++ ++ Yes, frozen section Foam FN _CC hMSC +++ ++ Textiles: Air FN _CC hMSC +++ ++ Foam FN _CC Schwann cells +++ ++ Foam IKVAV Schwann cells +++ ++ Foam FN _CC : IKVAV Schwann cells +++ ++ Textiles: Air FN _CC Schwann cells +++ ++ Textiles: Air IKVAV Schwann cells +++ ++ Textiles: Air FN _CC : IKVAV Schwann cells +++ ++ Foam FN _CC Min6m9 ++ ++ Yes, frozen section Foam 2R Min6m9 + ++ Foam FN: 2R Min6m9 + ++ Foam FN: 2R MIP +++ ++ Foam FN Human islet + Foam FN: 2R Human islet cell + Foam FN hESC ++ ++ film FN hESC ++ ++ Foam FN hIPS ++ ++ film FN hIPS ++ ++ film FN SMC ++ ++ film FN HUVEC ++ ++ Foam FN HUVEC ++ ++ *: HSkMC = human skeletal muscle satellite cells; HDMEC = human skin microvascular endothelial cells; HSMM = human skeletal muscle myoblast; HDFn = human skin fibroblast; HaCaT = human keratinocyte cell line; mEC = mouse endothelial cells; mMSC = mouse mesenchymal stem cells; hMSC = human mesenchymal stem cells; Min6m9 = pancreatic? -Cell; MIP = Islet from MIP-GFP mouse; hESC = human embryonic stem cells; hIPS = human induced pluripotent stem cells; SMC = smooth muscle cells; HUVEC = Human umbilical vein endothelial cells
**: + = 50-70%; ++ = 70-90%; +++ => 90%
***: + = cell increase in 1-7 days; ++ = cell increase at 1-14 days; +++ = cell growth in 1-21 days

Example

Example  One

Materials and methods

Production of recombinant spider web protein

The production of recombinant silk proteins in E. coli and the subsequent purification are essentially performed in accordance with the method of Hedhammar M et al . (Hedhammar M et al ., Biochemistry 47 (11): 3407-3417 (2008)) and Hedhammar M et al . (Hedhammar M et al ., Biomacromolecules 11: 953-959 (2010)).

Briefly, Escherichia coli BL21 (DE3) cells (Merck Biosciences) with an expression vector for the target protein were grown at 30 ° C on Luria-Bertani medium containing kanamycin at an OD ₆₀₀ of 0.8-1 , Isopropyl [beta] -D-thiogalactopyranoside, and further cultured for at least 2 hours. The cells were then harvested and resuspended in 20 mM Tris-HCl (pH 8.0) supplemented with lysozyme and DNase I. After complete dissolution, the supernatant from 15,000 g of centrifugation was loaded onto a column filled with nickel sepharose (GE Healthcare, Uppsala, Sweden). The column was extensively washed with 300 mM imidazole prior to elution of the bound protein. Fractions containing the target protein were collected and dialyzed against 20 mM Tris-HCl (pH 8.0). The target protein was released from the tag by proteolytic cleavage. To remove the HisTrxHis tag released, the cleavage mixture was loaded onto a second nickel-sepharose column and the passage was collected. Protein content was determined from the absorbance at 280 nm.

The resulting protein solution was purified from lipopolysaccharide (lps) as described by Hedhammar et al. (Hedhammar et al. , Biomacromolecules 11: 953-959 (2010)). The protein solution was sterile filtered (0.22 [mu] m) before use to prepare the support (film, foam, coating or fiber).

The recombinant spider mite protein was successfully expressed in E. coli and purified with similar yield and purity as the original 4RepCT.

Was used as a base for all proteins using the partial spider web protein 4RepCT (SEQ ID NO: 2). A functionalized version of 4RepCT with modified cell binding motifs from fibronectin, designated as FNcc-4RepCT (SEQ ID NO: 27) in the experimental section, was used for most experiments. 2RepRGD2RepCT (" 2R ", SEQ ID NO: 28) and 3RepRGD1RepCT (" 3R ", SEQ ID NO: 29), in which RGD peptides were inserted in the repeating portion, were used in some experiments using endocrine cells and other cells. Another version of GRKRK-4RepCT (SEQ ID NO: 30), in which the GRKRK peptide was inserted at the N-terminus, was used in some experiments using muscle satellite cells. Another version, IKVAV-4RepCT (SEQ ID NO: 31), in which an IKVAV peptide was inserted at the N-terminus, was used in some experiments using Schwann cells.

Cell culture

Mesenchymal stem cells (MSC)

(MMSC, Gibco) were cultured in DMEM F12 HAM supplemented with 10% fetal bovine serum (Mesenchymal Stem Cell Qualified, USDA Approved Regions, Gibco).

Transfection from Bone Marrow 8 Human mesenchymal stem cells (hMSCs) (Gibco) were cultured in complete StemPro MSC serum-free medium CTS (Gibco) containing 2 mM Glutamax in a culture flask coated with CELLstart (Gibco).

Endothelial cells (EC)

Mouse endothelial cells (Cell Biologics) were cultured in complete endothelial cell culture medium (PromoCell GmbH, Germany) at a passage number of 7-9.

Human dermal microvascular endothelial cells (HDMEC) (PromoCell GmbH, Germany) isolated from adult donor dermis were cultured in complete endothelial cell culture MV (PromoCell GmbH, Germany) in a culture flask coated with gelatin (Sigma Aldrich).

Fibroblast (HDFn)

A human skin fibroblast HDF (ECACC, Salisbury, UK) with a passage number of 8-11 was used. DMEM F12 ham supplemented with 5% FBS (Sigma) was replaced every 2-3 days.

Keratinocytes ( HaCaT )

HaCaT (human keratinocyte cell line, spontaneously transformed) was cultured in DMEM F12 ham supplemented with 5% FBS (Sigma). The medium was replaced every 2-3 days.

Human skeletal muscle satellite cells (Hsk)

Human skeletal muscle satellite cells, HskMSC (ScienCell Research Laboratories, Carlsbad, Calif., USA) and human skeletal muscle myoblasts (HSMM, Lonza, Belgium) were used in passages 2-6. SkMCM (ScienCell Research Laboratories), a skeletal muscle culture medium supplemented with skeletal muscle cell growth supplements, SkMCGS (ScienCell Research Laboratories) or SkGM-2 Bullet Kit (Lonza for HSMM), and 5% FBS (ScienCell Research Laboratories or Lonza, respectively) Replaced every 2 days.

Schwann cells

Schwann cells (3H Biomedical, Uppsala, Sweden) of passage number 2 to 6 were cultured in Schwann cell medium (SCM) supplemented with 5% FBS and Schwann cell growth supplements (SCGS, 3H Biomedical) and penicillin / streptomycin solution (3H Biomedical) , 3H Biomedical).

Endocrine cells

The pancreatic? -Cell MIN6m9 of the passage number 27-35 was incubated with? -Mercaptoethanol (50 μM), penicillin (100 U mL ^-1 ), streptomycin (100 μg mL ^-1 ), 10% heat- And cultured in DMEM supplemented with glucose (11 mM) (Gibco).

Islets from MIP-GFP mice, all raised in the animal core facility of the Karolinska Institutet, were isolated from the pancreas by injection of 1.2 mg / ml collagenase into the bile ducts. The pancreas was completely removed and placed in a flask containing the same concentration of collagenase as above. The flask was then placed in a 37 ° C water bath for 15 minutes. The islets were then washed and selected under stereoscopic microscopy. In order to disperse the islets in the cell, washed twice with PBS before the island-free Ca ^{2 +} and Mg ^{2 +,} and the cells were cultured at 37 ℃ for 5 minutes in Accutase (Gibco). Cells were counted and resuspended in RPMI 1640 supplemented with L-glutamine (2 mM), penicillin (100 U mL ^-1 ), streptomycin (100 ug mL ^-1 ), and 10% heat-inactivated fetal bovine serum 0.0 > (Gibco). ≪ / RTI >

Human islets were obtained from an inevitable excess of islets in the Nordic Network for clinical islet transplantation. Only donors who explicitly agreed to organ donations for scientific purposes were included. Written consent has been obtained from donors or donor associates through the Swedish National Board of Health and Welfare (Socialstyrelsen) for the donation of organs for medical and research purposes. The experimental procedure was carried out in accordance with an ethical license (license number 2011 / 14667-32) from the Ethical Committee for Human Research. HEPES (10 mM), L- glutamine (2 mM), gentamicin (50 mg ml ^-1), Hoon existing ^{(Fungizone, 0.25 mg ml -1,} Gibco) for human cells, ciprofloxacin (20 mg ml ^-1, Bayer Healthcare AG), nicotinamide (10 mM), and CMRL-1066 (ICN Biomedicals) supplemented with 10% heat-inactivated FBS.

Hepatocyte

The liver hepatocyte (liver cell) of rodents was transfected with hepatic enzymatic (25 mM Hepes, 1.2 mg / ml collagenase P in pH 7.4 HBSS buffer supplemented with 0,25% w / v BSA), collagen And then digested by continuous mechanical shaking at 37 캜 for 20 minutes, and then separated and cultured in RPMI-1640 medium supplemented with 10% FBS (Invitrogen).

Glioma-forming cell line

The glioma-forming cell line GL261 was cultured in DMEM (Invitrogen) containing 10% FBS while the medium was changed every 2-3 days.

Co-culture

HsK cells were cultured in SkMCM culture medium in co-culture with EC. In co-culture with MSC and EC, endocrine cells were cultured in RPMI 1640 medium (Gibco) at a ratio of 50:25:25, StemPro MSC serum-free medium CTS (Gibco) containing 2 mM Glutamax and endocrine cell medium MV (PromoCell GmbH, Germany ).

With integrated cells silk  Formation of support

Fiber formation

Silk proteins (0.5 - 3 mg) were mixed with 500 - 2 million cells in each culture medium in a total volume of 2-4 ml. Fiber formation with the cells was performed at room temperature with gentle shaking for 1-3 hours. The formed fibers were then washed with 1X PBS, transferred to a non-treated 12 or 24-well plate, and cultured in fresh medium (0.5 mL in 24-well plate or 1 mL in 12-well plate) Further maintained in water.

For fiber formation on the oil, one of 3-4 ml of FC40 (3M), HFE7100 or HFE7500 (Novec) oil was used.

For the pre-fabricated fibers, 70,000 cells were added to each piece of fiber (corresponding to 1/4 of that obtained in each tube), cultured in 96 wells for 1 hour and then seeded into 24 wells containing 1 ml of fresh medium I moved.

Foam  formation

A silk foam support was made from 20-40 [mu] l of silk protein (3 mg / mL) placed in the middle of the hydrophobic culture wells. Air was pipetted 30 times with 20 [mu] l protein drops. Cell suspensions (500,000-2,000,000 cells / ml) were prepared in each culture medium containing 25 mM Hepes but not serum, and then added dropwise (10-20 μl) either before or after the introduction of air bubbles, . Cell-containing foam plates were incubated in a cell incubator for 30-60 minutes before adding the appropriate cell culture medium.

Film formation

To remove aggregates, the silk protein (3 mg / mL) was thawed and centrifuged. 5 or 10 [mu] L of protein solution was added to the hydrophobic culture wells (Sarstedt suspension cells) to create liquid droplets on the surface of the well bottoms. The same volume of cell suspension (HDFn or HaCaT, 0.5 milj / mL, 1 milj / mL or 2 milj / mL) was then added to the protein droplets. The cell-containing film was incubated in a cell incubator for 30-60 minutes and then cultured in a LAF bench without a lid for 30 minutes (5 + 5 μL film) or 60 minutes (10 + 10 μL film) The medium was added. Culture was performed for 2 or 3 days followed by Live / Dead assay (Life Technologies).

3D with hepatocytes and glioma-forming cells Foam  formation

Recombinant spider-line proteins were used to prepare foams of 20 [mu] l protein (3 mg / mL), which were placed in the middle of the wells of a 24 well plate. Air was pipetted into 20 μl protein bubbles. The cell suspension (1 million cells / ml) was prepared in serum-free DMEM (Invitrogen) containing 25 mM Hepes. The final amount of 20,000 cells (20 [mu] l) from the prepared cell suspension was carefully placed on top of the foam with small droplets. The cell-containing foam was incubated in a cell incubator for 1 hour and then RPMI-1640 medium supplemented with 10% FBS (500 μl, Invitrogen) was further added.

silk  Analysis of cells in support

multiplication

Alamar Blue (Invitrogen, Stockholm, Sweden) was used to investigate cell viability and proliferation integrated into fibers and foams over a period of up to 21 days. AlamarBlue was diluted 1/10 in the appropriate cell culture medium, added to each well containing fiber or foam, and incubated in a cell incubator for 2 hours. After incubation, the supernatant was transferred to a new 96-well plate (Corning) and the OD was measured at 595 nm using a multimodal plate reader (ClarioStar, LabVision). The OD was plotted as fluorescence intensity per well. After the alamar blue culture and removal, the culture was continued using fresh complete medium.

Incubation of cell-containing silk scaffolds On days 3, 10 and 14, BrdU (Invitrogen) was added to a final concentration of 10 μM, incubated with BrdU for 20 hours, and then washed and fixed to produce frozen sections. DNA denaturation was performed on ice in 1 N HCl for 10 min, room temperature in 2 N HCl for 10 min, followed by 37 ° C for 20 min. Neutralization was performed immediately at room temperature in 0.1 M borate buffer pH 8.5 for 10 minutes. Samples were washed 3 x 5 min with PBS (pH 7.4) containing 0.1% Triton X-100 and blocked with PBS / 1% BSA for 15 min. The staining was performed using rdU-mouse monoclonal antibody (Clone MoBU-1) conjugated with Alexa-488 (Molecular Probes B35130) at 4 ug / mL in PBS / 1% BSA for 1 hour (or overnight at + . Control staining was performed with DAPI. Slides were mounted with a fluorescent mounting medium (Dako). Micrographs were taken at 10 × and 20 × magnification on a Nikon inverted fluorescence microscope.

Survival

Survival / death cell viability assays (Molecular Probes / Invitrogen, Stockholm, Sweden) were performed on cell-containing silk supports at selected endpoints after 7-21 days of incubation. After the silk support was washed with PBS, a mixture of calcein (1/2000) and EthD-1 (1/500) in PBS was added to the wells and incubated at room temperature for 30 minutes. The staining was then analyzed for live (green) and dead (red) cells using a fluorescence-based inverting microscope (Eclipse, Nikon, Sweden). Images were taken at a magnification of 10x on a selected plane of the support. Software NIS-elements were used to calculate the% viability (total amount of green cells / total cells × 100) in 3 equal zones per image.

Cell diffusion and attachment

After gently washing, the cell-containing silk support was fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, blocked with 1% bovine serum albumin (BSA, AppliChem) in PBS . Primary antibody mouse anti-human vinculin (Sigma V9131) was used at a concentration of 9.5 μg / ml in 1% BSA. The secondary antibody was cross-adsorbed, AlexaFlour 488 goat anti-mouse IgG (H + L) (Invitrogen) at 1: 500. The fibrous actin was detected using Phalloidin-AlexaFluor594 (Life Technologies) at 1:40. DAPI was used for nuclear staining. Slides were mounted on a fluorescent mounting medium (Dako, Copenhagen). The stained cells were analyzed at 4x and 10x magnification using an inverted microscope (Nikon Eclipse Ti).

Cell distribution and morphology

At the end, the cell-containing silk scaffold was fixed in 4% paraformaldehyde for 15-30 minutes, washed and incubated in 20% sucrose until embedded in Tissue-Tek (Sakura, Japan) The cryostat was cut into 12-25 μm thick sections. The morphology of the selected sections after standard hematoxylin and eosin (HE) staining for frozen tissues was evaluated.

differentiation

Selected sections of the cell-containing silk support were permeabilized with 0.5% Triton X-100 for 5 minutes, blocked with 5% normal goat serum in PBS for 30 minutes at room temperature and then treated with desmin (Anti-Des, Prestige Antibodies, Atlas Antibodies, Sigma Aldrich, 1: 200). Next, the fiber sections were probed with a secondary antibody obtained from goat for rabbits conjugated to Alexa 488 (Molecular Probes, 1: 1000).

Collagen production

The sections were blocked with 1% BSA in PBS, stained with mouse anti-collagen type I (clone COL-1, SigmaAldrich) to 3.5 mg / mL in 1% BSA and then stained with AlexaFluor488 goat anti-mouse IgG antibody (Invitrogen) Lt; / RTI > DAPI was used for nuclear staining. Slides were mounted with a fluorescent mounting medium (Dako). Microscopic pictures were taken at 10x with a Nikon inverted fluorescence microscope.

Insulin production and secretion

The silk foam support with clusters of endocrine cells was washed with PBS, fixed with 1% paraformaldehyde, and permeabilized with PBS containing 0.3% Triton x 100 for 15 minutes. Blocking was performed with 6% fetal bovine serum (FCS) in PBS containing 0.1% Tween for 1 hour at room temperature (RT). Samples were then incubated with anti-insulin antibody (guinea-pig anti-insulin, 1: 1000, Dako), rabbit anti-human CD44 (1: 100) and / or mouse anti- human CD31 (1: 100, BD Pharmingen) Lt; RTI ID = 0.0 > 4 C. < / RTI > The next day samples were probed with secondary antibodies obtained from goat (Molecular Probes, 1: 1000) against rabbits and mice conjugated with Alexa488 conjugated guinea-pig and Alexa 594.

Endocrine cell clusters from MIP-GFP transgenic mice were incubated with hMSC and HDMEC in foams consisting of a mixture of 2R and FN proteins in 24-well plates for 7 days. The foam was placed gently on top of a 0.5 ml column filled with Bio-Gel P4 polyacrylamide beads (Bio-Rad). The kinetics of insulin release was observed at 37 ° C by Hepes buffer with 3 mM glucose as the basic condition and with Hepes buffer with 11 mM as the stimulating glucose concentration for insulin release followed by perifusion of the clusters with 25 mM KCl . The flow rate was 40 ml / min, and the 2 min fraction was collected and analyzed for insulin using the insulin assay HTRF kit (Cisbio).

Cell silk  Mechanical analysis of supports

The stress versus strain (% length extension) of the cell-containing fibers was measured using a ramp force of 0.2 N / min with a Zwick / Roell material tester custom-made under physiological similar conditions (37 ° C, 1 × PBS) Respectively. The fiber ends were mounted with a sample gripper. Macroscopic defects or those that were obviously damaged during mechanical testing were excluded. The initial section of the circular form of the fiber was used for the stress calculation.

cell silk  Transplantation of structures and in vivo imaging

Implants were essentially performed using the same procedure as described previously in Speier S, et al . (Speier S, et al . Nat Protoc. 3: 1278-1286 (2008)). The cell-containing silk matrix was cut into smaller pieces (~50 um), placed in a sterile culture medium, and placed in a 27 gauge eye (1 ml) connected to a 1 ml Hamilton syringe (Hamilton) via a 0.4-mm polyethylene tube eye cannula (manufactured by retrofitting a patch-clamped glass capillary with a blunt end). B6 Albino A ++ (C57BL / 6NTac-Atm1.1Arte Tyrtm1Arte, Taconic, Cologne, Germany) purchased from Jackson Laboratory (Bar Harbor, Maine, USA) was anesthetized with 2% isoflurane (vol / vol) Respectively. When the cannula was stably inserted into the anterior chamber, the implant was slowly injected into the anterior chamber with the smallest possible volume of sterile saline solution and fixed on the iris. Analgesia was induced after surgical treatment with buprenorphine (0.05 - 0.1 mg / kg sc).

In vivo imaging of the scaffolds in the anterior chamber of the implanted animals was essentially performed as previously reported in Speier S, et al . (Speier S, et al . Nat Protoc. 3: 1278-1286 (2008)). Briefly, the mice were anesthetized with a 2% isoflurane air mixture and placed on a heating pad, and then the head was held in the head holder. Viscotears (Novartis) were used as an immersion liquid between the eyes and the object, with the eyes gently fixed by pulling back the eyelids gently. Scanning speed and laser intensity were adjusted to avoid cell damage to the mouse eye.

result

With integrated cells silk  Formation of support

Figure 3 schematically shows the formation of a silk support with integrated cells.

Fiber formation

3A schematically shows the formation of cell-containing silk fibers. The silk protein was mixed with the suspended cells in the medium (I). Silk protein is assembled into a fibrous mat with integrated cells at the air-liquid interface (II), while gently shaking during 1 to 3 hours of incubation. Cell-containing silk fibers are then easily recovered and placed in culture wells (III).

The gentle agitation of the silk protein solution mixed with the cells in the tube culture medium resulted in the formation of visible fibers within 20 minutes, i.e. within the same time as for the silk protein alone. After allowing fiber formation to last for 1-3 hours, the fiber bundles were transferred to cell culture wells containing fresh medium. Visually, the cell-containing fibers looked very similar to normal silk fiber bundles on day 1 (FIG. 3A), but continued to increase in thickness throughout the incubation period. For some cell types, such as fibroblasts and skeletal muscle satellite cells, smaller fiber bundles typically have been rounded after several days of incubation. This could be avoided by mounting them as elongated fiber bundles between two anchor points using inserts in the wells.

Due to sedimentation, significant cell fractions were found at the bottom of the tube during fiber formation. To avoid such cell loss, an inverted configuration with a dense oil phase under the silk / cell solution was developed. Using this approach, cell-containing fibers were formed at the buffer: oil interface where cells were trapped instead of forming at the air: buffer interface. Using this method, higher cell densities in the fibers were obtained, though the cost was higher due to the uneven shape.

Foam formation

Figure 3B schematically illustrates the formation of cell-containing silk foams. The silk protein solution containing cells was transformed into a wet foam by gently introducing air (I). After pre-incubation for 30-60 minutes additional culture medium was added to cover the foam (II). Cellular silk foams can then be cultured in wells (III). Scale bar = 1 mm.

The smooth introduction of air bubbles into the mixture of silk protein solution and cells in the culture medium resulted in an expanding foam structure, similar to that made with silk proteins alone (Fig. 3B). When a fresh cell culture medium is added after a pre-incubation period of 30 to 60 minutes, the foams are held together as a consistent three-dimensional structure. Throughout the incubation period, the foam gradually becomes white and loses its transparency.

Film formation

Figure 3C schematically shows the formation of a cell-containing silk film. The silk protein solution was added dropwise into the culture well, and the cells suspended in the medium were added dropwise one by one (I). After 30-60 minutes pre-culture, additional culture medium was added to cover the film (II). Cell-containing silk films could then be cultured in wells and L / D staining performed (III). Left: 4 × magnification of HDFn (20,000 cells / film) after 2 days, and right: 4 × magnification of HaCaT (10,000 cells / film) after 3 days.

When pre-incubated for 30-60 minutes before the new culture medium is added, the cells are kept together as a coherent film by addition of cells in the culture medium into defined drops of silk protein solution. Depending on the amount of cells added, the cell-containing film reaches a dense state within 1-3 days of incubation.

The cell silk  The ability to multiply within the support Keep

The measurement of cell proliferation (using the Alarama blue cell viability assay) confirmed the growth profile of cells proliferating in all the foams, fibers and films. Figure 4 shows the metabolic activity of cells in a silk support. Figure 4A shows representative growth profiles of individual silk fiber bundles containing different cell types (mMSC, mEC, HDFn, Hsk) measured using the alamarv blue viability assay. Figure 4B shows representative growth profiles in individual silk foams containing different cell types (mMSC, mEC, HaCaT, MIN6m9) measured using the alamarv survival assay.

The amplitude of the signal varied between samples of fiber bundles, possibly reflecting an uneven distribution of the captured cells. This may be avoided, in part, by using higher cell densities and faster processing before fiber formation is initiated. For the foam and film formats, the growth profile was more reproducible between samples, possibly due to the fact that all the cells added to it are trapped directly within the support.

The slope of the growth curve was affected by both cell density and cell type used. Typically, a slower initial phase can be observed, and then a more steep curve can be observed. Samples that reached a high plateau two weeks later typically contained a dense cell layer, as could be confirmed by cytotoxicity (see below).

In order to investigate whether the cells integrated in the silk support (as well as on the surface cells) were cleaved and proliferated, we also performed BrdU assays. By adding BrdU to the medium during the last 20 hours before immobilization, cells that cause cell division will integrate BrdU molecules into their genomes during DNA-synthesis. These BrdU molecules can then be detected by immunofluorescence. In this way, proliferating cells deep within silk fibers could be identified at all time points examined (days 4, 11, and 15). The proportion of dividing cells was higher at baseline and decreased during the incubation period (80% at 4 days, 50% at 11 days, 25% at 15 days), which is normal for in vitro cultures where cells become dense.

Most cells silk  Survival in the support It is possible

The viability of the cells in the silk support was analyzed under a microscope using a two-color fluorescence viability assay that simultaneously stained live (green) and dead (red) cells.

Figure 5 shows cell viability in a silk support.

A) Survival staining of various types of cells in cellular silk fibers (10x).

B) Survival staining of various types of cells in cell-containing silk foam (10x).

C) Cell viability in the fiber.

D) Cell viability in the foam.

Although visually apparent, the support looked somewhat thicker than usual silk material, but under fluorescence microscopy it became clear that the sample contained a significant amount of cells, many of which were alive (FIG. 5). Survival in all fibers exceeded 80% (Fig. 5C), well over 90% for all foam supports (Fig. 5D). In the case of the film, viability was largely dependent on the amount of cells added, and viability was 80-90% if the cells were added in excess of the number of cells that could enter the dense layer (data not shown).

The cell silk  Through local attachment in the support Diffused Attached

The ability of cells to elongate and diffuse within the silk support was evaluated by staining for stress fibers (via actin filaments). Figure 6 shows the diffusion of cells in a silk support. Figure 6A shows f-actin staining (left) of HDFn cells in the fiber and dapi (rounded spots indicate nuclei) and f-actin staining (right) of mMSCs in the foam (10x). Figure 6B shows f-actin and binuclein (light spot) staining of HDFn (left) and HDMEC (right) in the fiber.

In the fiber format, the cells were found along the bundle of fibers and mostly elongated cell shapes (Fig. 6A, left). In the foam format, cells were typically stretched and found to extend all the way between silk structures (Fig. 6A, right). Well-organized actin stress fibers were found in most cells in all fibers and foams.

Cell attachment through the formation of focal adhesion was analyzed after staining for F-actin, often associated with vinculin, one of the major components of the local adhesion complex located near the cell membrane. Thus, co-staining of F-actin and vaculin is involved in integrins and is a signal of well-established binding of cells to the support. Within cell-containing fibers, it was possible to distinguish local attachment points as bright spots at the edges of elongated cells (Fig. 6B). Within the foam support, the cells were randomly distributed in three dimensions, and although they could sense a clear signal from the vinculin staining (data not shown), the distinction of local attachment points was complex.

The cell silk  Throughout the support Distributed

In order to confirm whether the cells are well distributed in the silk support, the present inventors performed the preparation of frozen sections and H / E staining to determine the location of the cells. The fibers were cut in both the longitudinal direction and the width direction of the fiber axis (Fig. 7A). Cells were visible throughout the fiber, but some areas were more dense than others. According to the viability test results, the foam support had more dense cells throughout the material (Fig. 7B).

Figure 7 shows the distribution of cells in a silk support. Figure 7A shows H / E staining of the longitudinal (left) and transverse (right) frozen sections of silk fibers with HDFn cells. Figure 7B shows H / E staining of frozen sections of HaCaT (left) and mMSC (right) cellular silk foams. Dark spots represent nuclei.

With cells silk  The support is mechanically Be stable

The cell-containing silk supports were stable enough to handle throughout the incubation period and analysis procedure and were similar to conventional silk supports in terms of flexibility under humid conditions. Tension tests of cell-containing fibers were performed in pre-warmed physiological buffer to relate mechanical properties as compared to native tissue (Fig. 8). After the initial elastic phase, the deformation zone was reached and the fiber was extended to about twice its initial length.

Figure 8 shows the mechanical properties of silk fibers with cells by the stress strain curves of two representative silk fibers containing fibroblasts (HDFn) cultured for two weeks.

Fibroblasts silk  Collagen in the support Generate

We examined whether fibroblasts produced collagen type I when grown in a variety of support types, as a first step to confirm that the cells retained their main function during incubation in a silk support. By staining intracellular collagen type I, it was clear that most cells, even in the fiber or foam, produced collagen.

Figure 9 shows immunofluorescence staining of collagen type I. Silk scaffolds with fibroblasts were incubated for 2 weeks before staining with collagen type I specific antibodies for detection of native spiral collagen type I. Specific antibodies detect both intracellular and extracellular collagen. Round spots indicate Dapi staining of the nucleus.

silk  Within the support, cells can be differentiated have

To determine if cells could be differentiated in the silk support, the fibers with human skeletal muscle satellite cells were transferred to DMEM culture medium to promote differentiation. Desmin staining was used to visualize the formation of myotubes (Fig. 10).

Figure 10 shows immunofluorescence staining of formed canals. Hsk cells were incubated for 2 weeks, then maintained in differentiation medium for 2 weeks and stained with Desmin. Round spots indicate Dapi staining of the nucleus.

Several types of cells silk  Can be co-cultured in a support have

Most unique tissue types consist of several types of cells that are organized together in a complex three-dimensional array with the extracellular matrix surrounding the cells. Therefore, in order to replicate this in an artificial tissue structure, it is important to achieve co-culture in a support. Using the methods described herein for the formation of cell containing silk supports, it is substantially very easy to combine several cell types as long as they can be cultured in similar media.

The present inventors herein demonstrated an example of co-culture with silk fibers using human skeletal muscle satellite cells and endothelial cells (Fig. 11A). Endothelial cells have been found to be distributed along with some local clusters within the fibers, possibly indicating an initial state of angiogenesis.

As an example of co-cultivation in silk foam, we bound mesenchymal stem cells supporting endocrine cells and endothelial cells (FIG. 11B).

Figure 11 shows the presence of various cell types co-cultured in a silk support. Figure 11A shows cross sections of silk fibers co-cultured and immunostained for EC (top) and Hsk cells (bottom). Figure 11B shows silk foam co-cultured and immunostained for MIP (top) and MSC (bottom).

silk  Endocrine cells within the support are functional Keep

Endocrine pancreatic islets found in the pancreas, often referred to as the islets of Langerhans, are typical examples of cells that require appropriate neighboring cells as well as physical three dimensional support to maintain functionality.

Figure 12 shows that an islet-like cluster is functional in a silk support. Figure 12A shows insulin staining of endocrine cells and their clusters in silk foam. Solutions of dispersed endocrine cells, recovered by dissociation of isolated islet cells, tend to aggregate into islet-like shapes when cultured in silk foams. The staining for insulin confirms that not only the cluster but also single cells maintain the ability to produce insulin in the silk foam (Fig. 12A).

To further illustrate whether insulin-like clusters formed in the silk foam were functional, that is, whether insulin was produced only when stimulated, the amount of insulin was measured after stimulation with physiological concentrations of glucose. Figure 12B shows a representative curve of dynamic insulin release after peripheral perfusion of islet-like clusters in silk foam. The insulin values were normalized to dsDNA and the insulin values of the charts were expressed as% of basal levels. To imitate physiological stimuli as much as possible, the clusters were dynamically stimulated with increasing glucose concentrations. Silk foams containing islet-like clusters were pumped through buffers with varying glucose concentrations and placed in columns that were kinematically peripherally perfused. Thus, an increase in insulin release after stimulation with a high concentration (11 mM) of glucose could be measured, which was reversed when the glucose concentration returned to the basal level (3 mM) (Figure 12B). In addition, clusters within the silk foam responded to subsequent KCl stimulation.

With cells silk  In vivo imaging of scaffolds

Next, we examined how the cell-containing silk support could be persistent in vivo. Cells were first cultured in fiber and foam, respectively, and transplanted into the anterior chamber of the mouse one week later. The open part of the eye was utilized to evaluate the silk support using a camera (Fig. 13, left), and was used to evaluate cells therein (in vivo traced) using confocal microscopy (Fig. 13). The macroscopic appearance of the silk supports was similar in vivo over 4 weeks, while the distribution and amount of cells changed slowly, possibly due to cell migration as well as degradation.

Figure 13 shows an in vivo image of a silk support with cells. The left side shows photographs of cell-containing (mMSC) fibers (white) implanted in the anterior chamber. On the right is a representative confocal microscope picture of in vivo traced cells (mMSC) in silk fibers after 1, 2 and 4 weeks in vivo.

Integration of cells silk  At the time of addition to the protein Depend on

An alternative manufacturing protocol was investigated to determine how the cells were distributed within the silk support according to the stage in which the cells were added during the manufacturing process.

Fiber formation occurs at the hydrophilic / hydrophobic interface in the tube being cultured during gentle shaking. In order to maintain the sterilization conditions, the tubes should be closed during incubation, which has two options for cell addition: addition to the silk protein solution before initiation of fiber formation, or addition to the top of the formed fiber after recombination and well cultivation There is only one thing to do. Since the fibers are formed as bundles, penetration of some cells is possible even when cells are added after fiber formation (Fig. 14, right column). However, if the cells were added to the silk protein solution prior to fiber formation, the cells would be more evenly distributed within the fibers (Fig. 14, left column).

Figure 14 shows the distribution of cells in the silk fibers. H / E staining of frozen sections of silk fibers containing HDFn (upper row) and EC (lower row) added before (left column) or after (right column) Dark spots represent cell nuclei.

Foam formation is achieved by gently introducing air bubbles into the silk protein solution. The silk support slowly solidifies at the interface of each air bubble. If cells (in the medium) are added directly to the silk protein solution prior to introduction of air bubbles, the cells are evenly distributed throughout the silk foam. If the cells are added dropwise after the formation of the foam, the cells in the medium slowly spread through one foam structure where the foam is still wet; The more cells added earlier, the more evenly distributed. If cells are added to a dry foam, the foam structure is partially broken down to form a thinner, net-like silk structure.

A foam support containing cells added at different times was stained with f-actin (to visualize the cells) and photographed using an inverted fluorescence microscope. The distinct and distinct cells seen in several z-planes of all analyzed foam supports were cells added (0-90 min) before drying (Table 3). In the case of a dried foam support before addition of cells, it was only possible to distinguish cells from one z-plane.

Analysis of cell-added silk foam supports at various time points When the endothelial cells were added
(minute) Number of z-planes with different cells
(Fluorescence microscope) On the floor
Cell number
(H / E staining of frozen sections) When fibroblasts are added
(minute) Number of z-planes with different cells
(Fluorescence microscope) On the floor
Cell number
(H / E staining of frozen sections) 0 3 10-15 0 3 6-8 10 3 10 10 3 n.a. 60 3 5 60 2 3-5 90 4 n.a. 90 3 3-5 240 (dry) One n.a. 240 (dry) One 1-2

The foam support was further examined by staining with frozen sections (on the side) and H / E. For all the foam supports analyzed, the support was added with cells (0-90 min) before drying and the support had a poofy appearance with several layers of cells (Fig. 15, left column). The foam support, which was allowed to dry before the cells were added, was located as a thin and compacted line where most cells were either one or a maximum of two cell layers (Fig. 15, right column).

15 shows the cell distribution in the silk foam. H / E staining of frozen sections of silk foam with HDFn (top row) and EC (bottom row) added to the silk protein solution at time 0 (left column) or after 240 min (right column)

Example  2. With an alternative C-terminal domain Mini-speed lane To foam  Integration of cells

The preparation and purification of the silk protein FN _cc -RepCT _MiSp (SEQ ID NO: 69) was carried out as described in Example 1. CT _MiSp (SEQ ID NO: 68) is a bovine spider web protein derived from Aranaeus ventricosus .

Primary endothelial cells (HUVEC, PromoCell) from capillaries of human origin were cultured in endothelial cell growth medium MV2 (PromoCell) containing bovine serum albumin (FBS, 5%). Cells of passage number 6 were used.

Silk foam supports were prepared with 20-40 [mu] l of silk protein (3 mg / mL) placed in the midpoint of the hydrophobic culture wells. Air was pipetted 30 times into 20 μl protein bubbles. Cell suspensions (500,000-2,000,000 cells / ml) were prepared in each culture medium containing 25 mM Hepes but no serum and added dropwise directly after introduction of air bubbles (10-20 μl ). Cell-containing foam plates were incubated in a cell incubator for 30-60 minutes and then the appropriate cell culture medium was added.

The viability and proliferation of the integrated cells was examined using Allama Blue (Invitrogen, Stockholm, Sweden). Alamar blue was diluted 1/10 in the appropriate cell culture medium, added to each well containing the foam, and then incubated in a cell incubator for 2 hours. After incubation, the supernatant was transferred to a new 96-well plate (Corning) and OD was measured at 595 nm using a multimodal plate reader (ClarioStar, LabVision). OD was plotted as fluorescence intensity per well. The culture was then continued with fresh complete medium after the alamar blue incubation and removal.

Survival / death cell viability assays (Molecular Probes / Invitrogen, Stockholm, Sweden) were performed 8 days after incubation in cell-containing silk foams. After washing the silk support with PBS, a mixture of calcein (1/2000) and EthD-1 (1/500) in PBS was added to the wells and incubated at room temperature for 30 minutes. The staining was then analyzed for live (green) and / or dead (red) cells by fluorescence inversion microscopy (Eclipse, Nikon, Sweden). Images were taken at 4x magnification on selected planes of the support.

Figure 16 shows the growth curves (20,000 HUVEC / well) of cells proliferating in the foam of FN _cc -RepCT _MiSp (SEQ ID NO: 69; filled diamond) and the corresponding FN _cc -RepCT _MaSp n = 3, SEM), and similar proliferation was confirmed.

Figure 17 shows live cell staining at the end of culture (day 8) and shows the survival cells integrated in both the FN _cc -RepCT _MiSp (left panel) and the FN _cc -RepCT _MaSp (right panel) foam (4x magnification) .

Example  3. Silkworm Springbix Mori Bombyx mori) From silk  Integration of cells within the matrix of fibroin

The scraps of cocoons from B. mori were removed in 0.02 M sodium carbonate boiling, washed appropriately with distilled water, and then dried overnight at room temperature. The rubber component was then removed and the dried silk was dissolved in 9.3 M LiBr and dialyzed for 3 days using a dialysis membrane (MWCO 12 kDa) while continuously exchanging water for Milli-Q water.

For fiber formation, fibroin protein (0.5 - 10 mg) was mixed with 500,000 - 2 million cells in each culture medium in a total volume of 4 ml. The cells were allowed to undergo fiber formation with gentle shaking for 1 to 24 hours at room temperature. The formed fibers were then washed with 1X PBS and transferred to a non-treated 24-well plate followed by continued culture by adding fresh medium.

For foam formation, 20-40 μl fibroin protein (3 mg / ml) was placed in the middle of the hydrophobic culture wells. The air was pipetted 30 times into 20 μl protein drops. Cell suspensions (500,000-2,000,000 cells / ml) were prepared in each culture medium containing 25 mM Hepes but no serum and added dropwise before or after introducing air bubbles (10-20 / RTI > The plates were incubated in a cell incubator for 30-60 minutes and then the appropriate cell culture medium was added.

For film formation, 5 or 10 [mu] L fibroin protein solution (3 mg / mL) was added to hydrophobic culture wells (Sarstedt suspension cells) to create droplets on the well bottom surface. An equal volume of cell suspension was then added to the protein droplets. The cell-containing film was incubated in a cell incubator for 30-60 minutes and incubated in a capless LAF bench for 30 minutes before adding 1 mL of culture medium.

Cells were treated and cultured as described in Example 1. Alarama blue and survival / death viability assays were performed as described in Example 2. < RTI ID = 0.0 >

Figure 18 shows the growth of proliferating cells (hDF) in the fibers of B. mori silk fibroin (open triangle, dashed line) compared to the corresponding fibers of FN _cc -RepCT (SEQ ID NO: 27; Curve. Figure 19 shows staining of live fibroblasts (HDFn, ECACC, P7; scale bar 250 [mu] m) integrated into the fibers of B. mori silk fibroin and also confirms the presence of viable cells on day 15.

The presence of viable HUVEC in the foam of B. mori silk fibroin was determined 19 days after incubation (data not shown).

Figure 20 shows the effect of increasing the number of proliferating cells in a film of B. mori silk fibroin (" BM ", dark diamond type) compared to the corresponding film of FN _cc -RepCT (SEQ ID NO: 27; HUVEC) (n = 6, SEM; (A): 10,000 HUVEC / well; (B): 3,000 HUVEC / well). Survival staining further confirms the presence of viable cells in both types of films at day 8 (data not shown).

Example  4 - Human pluripotent stem cells ( hPSCs ) Integrated silk  Preparation of support

Foam formation

20 μl droplets of FN _cc -RepCT (SEQ ID NO: 27; 3 mg / ml) and laminin 521 (BioLamina, at a final concentration of 10 μg / ml) were placed in the center of the hydrophobic culture wells. Using a pipette set at 40 μl, pipetting air into the droplet quickly by pipetting up and down with 20 strokes, creating a dense wet foam. Within the Essential 8 ^TM medium (Life Technologies), 50,000 hPSCs, typically at a concentration of 10,000 cells / μl, were immediately introduced into the foam by another 10 strokes to disperse the cells throughout the 3D structure. Cell-containing foams were then stabilized in a 37 ° C incubator for 20 minutes and then added to a 24-well plate, 1 ml of Essential 8 ^™ containing the ROCK inhibitor Y27632, 10 μM. The following day fresh culture medium was added without ROCK inhibitor, and the medium was changed daily.

Film formation

A film was prepared by adding 10-20 μl of FN _cc -RepCT (SEQ ID NO: 27; 3 mg / ml) and laminin to the center of the hydrophobic well. The silk solution is formed into a desired shape and size using a pipette tip, and typically 30,000 to 50,000 hPSCs (at least 10,000 cells / μl concentration) are added by gently dripping to the center of the silk protein, . The film was then stabilized for 20 to 40 minutes in a cell incubator at 37 DEG C, depending on the size of the film, and 0.5 mL of the ROCK inhibitor, 10 [mu] M (suitable for 24-well plates) of Essential 8 ^TM medium was added . The following day fresh culture medium was added without ROCK inhibitor, and the medium was changed daily. The PSC integrated into the silk disk was easily monitored by bright field microscopy and the start of differentiation was determined when the cells reached a dense state for the selected protocol.

Foam  And the film PSC  Immunological staining for

Immunocytochemistry was performed at specific time points after the cells were integrated into the silk. The silk support was washed once with PBS and then 4% paraformaldehyde was added for 15 minutes. After permeabilization in PBS containing 0.1% Triton X-100 for 15 minutes, it was then blocked with 10% donkey serum (Jackson ImmunoResearch). Primary antibodies were incubated overnight at 4 ° C in PBS with 0.1% Tween-20 (PBS-T) and 5% serum. The secondary antibody was incubated in PBS-T and 5% serum at room temperature for 1 hour. Nuclei were counterstained with DAPI (Sigma) and incubated for 30 min. Samples were washed three times with PBS-T between each culture.

Primary antibody used: polyclonal goat anti-Naanog, 1:50 dilution (R & D), polyclonal rabbit anti-laminin, 1: 200 dilution (Abcam).

Secondary antibodies used: donkey anti-rabbit 688 (Abcam) and donkey anti-goat 488 (Jackson ImmunoReasech), both diluted 1: 1000.

Samples were imaged using a Leica DMI 6000 B microscope and image software ImageJ.

Figure 21 shows the incubation of PSC integrated in silk foam and film:

(A) Exemplary photomicrographs of foams and films of FN _cc -RepCT (SEQ ID NO: 27) with laminin 521 (LN521) at 24 hours after the injection of 50,000 human iPS C5. The cell distribution was visualized by nuclear DAPI staining (blue). The scale bar represents 1000 μm.

(B) Human embryonic cell HS980 proliferated well at 72 hours after incorporation into the foam (top panel) and film (bottom panel) of FN _cc -RepCT (SEQ ID NO: 27) as revealed by ICC, Respectively. BF is bright field. The laminin-coated silk was visualized in green by anti-laminin antibody (Abcam) and universal in red by anti-nanog (R & D). The nuclei were counterstained with DAPI (blue). The scale bar represents 200 μm.

(C) Representative photographs of iPS C5 cells that proliferate in FN _cc -RepCT (SEQ ID NO: 27) foam and film at 72 hours post-seal, visualized with a bright field microscope.

Conclusions: Human pluripotent stem cells (hPSCs) such as embryonic stem cells (ESC) and induced pluripotent cells (iPS) survive and proliferate well after integration into the foam and film of silk proteins.

Example  5 - Efficient Seeding  As a method silk  Integration of cells into film

Two different cell types were tested: smooth muscle cells (human coronary artery, Gibco) and human umbilical vein endothelial cells (Promocell). The cells suspended in each culture medium were mixed with FN _cc -RepCT (SEQ ID NO: 27; 3 mg / ml) 1: 1 and then cultured in wells (uncoated, pre-coated with gelatin, WT ", SEQ ID NO: 2) or FN _cc -RepCT ("FN", SEQ ID NO: 27).

The amount of cells attached within 30 min was analyzed after three subsequent washings prior to fixation and staining with crystal violet. The upper row in Figure 22 shows the absorbance from the crystal violet stained cells after being dissolved from the state attached to the culture well. When seeded into the silk film, considerably more cells adhered to the uncoated wells. 22, and the lower row shows a micrograph of the stained cells. The shape of the attached cells confirms proper attachment and diffusion.

It has been concluded that the formation of a film with integrated cells provides high seeding efficiency, producing rapidly and viably adherent cells.

Example  6 - silk  Differentiation of stem cells integrated into supporter

Fibers and foams were prepared from FN _cc -RepCT (SEQ ID NO: 27) with integrated human mesenchymal stem cell (hMSC) as described in Example 1.

(A) Fat production or osteogenesis differentiation

Macrostructures with integrated hMSC cells were treated with adipogenic or osteogenic differentiation medium (PromoCell) after 7 days of incubation. The medium was changed every 3 days until day 14. The samples were then fixed and stained with lipid markers, such as Red Oil O (Sigma Aldrich) for fat, and Algirin Red S (Sigma Aldrich), an osteogenic marker for bone, all according to standard protocols.

The upper row of FIG. 23 shows that the hMSCs differentiated into fat-producing strains contain lipid lipids (N = 2, n = 4) with red oil staining of the foam (left) and fiber (right). Scale bar = 100 μm. Illustrations are photographs of the follicles (differentiated (left) and undifferentiated (right), scaling bar = 6.6 mm), and fibers (unstained (left) and red oil stained (right), scaling bar 1 mm) . The lower line in Fig. 23 shows the difference in the calcium content (alizarin red S (red)) of the foam (upper left side, scale bar = 100 mu m) and fibers (upper right side, (N = 2, n = 4). ≪ / RTI > The illustrations are shown in Fig. 1 (left) and undifferentiated (right), scaled bar = 6.6 mm) and fibers (unstained (left) and alizarin red S stained (right), scaled bar = 1 mm) .

Lipid droplets were found in whole silk foams and fibers with integrated cells treated with adipocyte-derived medium (Fig. 23, upper row). Calcium was found to be deposited on the entire support treated with the osteoblast-derived medium and accumulated in the innermost portion of the fiber (Fig. 23, bottom row).

(B) Neurogenesis

Macro constructs with integrated hMSC cells were cultured for 3 days followed by dual-SMAD inhibition (Noggin and SB431542) for 7 days. This protocol produces neural progenitor cells. Then, the medium was replaced with neural progenitor differentiation medium, culture was continued for 14 days, and neural differentiation markers? III tub, MAP2 and GAD1 were then analyzed by RT-qPCR.

Figure 24 shows the relative gene expression analyzed by RT-qPCR of the neural differentiation markers beta III tub, MAP2 and GADl on days 0 and 21. All data represent mean ± SD for 5 independent cultures (n = 5).

It was concluded that human mesenchymal stem cells can be differentiated in silk supports. Successful differentiation could be confirmed after fixation, and as a lipid marker for fat and as an osteogenic marker for bone. Successful differentiation could also be confirmed after RT-qPCR analysis of neurogenic markers.

Example  7 - silk  Cell diffusion after integration into supporter

To investigate the effect of the fibrillar silk network on cell proliferation, the macro integrated structure in which the cells were integrated was prepared from FN _cc -RepCT (SEQ ID NO: 27) as described in Example 1.

For comparison, the same sample cell types were seeded into a hydrogel (NovaMatrix) of alginate with covalently associated RGD motifs. RGD alginate is prepared together with the cells as a 2% mixture in cell culture medium and immersed in CaCl ₂ (100 mM) to induce gelation.

High resolution three-dimensional photographs of the native hydration state of the silk and hydrogel supports with integrated cells were collected using a confocal reflex microscope.

Attachment and diffusion of integrated cells in silk and hydrogel supports were evaluated using a laser scanning confocal microscope. An inverted system equipped with a fluorescence and relative ratio was used to visualize both cells and material.

Immunohistochemistry can be used to identify the critical components of various stages of attachment (such as local complexes, local attachment, fibril attachment, and 3-dimensional attachment) at specific time points (eg, integrin, Actin).

                         SEQUENCE LISTING <110> Spiber Technologies AB   <120> Integrated cells <130> PC-21085957 <150> EP 16155494 <151> 2016-02-12 <150> EP 16194431 <151> 2016-10-18 <160> 69 <170> PatentIn version 3.5 <210> 1 <211> 789 <212> DNA <213> Euprosthenops australis <400> 1 ggtccgaatt caggtcaagg aggatatggt ggactaggtc aaggagggta tggacaaggt 60 gcaggaagtt ctgcagccgc tgccgccgcc gcagcagccg ccgcagcagg tggacaaggt 120 ggacaaggtc aaggaggata tggacaaggt tcaggaggtt ctgcagccgc cgccgccgcc 180 gcagcagcag cagcagctgc agcagctgga cgaggtcaag gaggatatgg ccaaggttct 240 ggaggtaatg ctgctgccgc agccgctgcc gccgccgccg ccgctgcagc agccggacag 300 ggaggtcaag gtggatatgg tagacaaagc caaggtgctg gttccgctgc tgctgctgct 360 gctgctgctg ccgctgctgc tgctgcagga tctggacaag gtggatacgg tggacaaggt 420 caaggaggtt atggtcagag tagtgcttct gcttcagctg ctgcgtcagc tgctagtact 480 gtagctaatt cggtgagtcg cctctcatcg ccttccgcag tatctcgagt ttcttcagca 540 gtttctagct tggtttcaaa tggtcaagtg aatatggcag cgttacctaa tatcatttcc 600 aacatttctt cttctgtcag tgcatctgct cctggtgctt ctggatgtga ggtcatagtg 660 caagctctac tcgaagtcat cactgctctt gttcaaatcg ttagttcttc tagtgttgga 720 tatattaatc catctgctgt gaaccaaatt actaatgttg ttgctaatgc catggctcaa 780 gtaatgggc 789 <210> 2 <211> 263 <212> PRT <213> Euprosthenops australis <220> <221> DOMAIN &Lt; 222 > (1) .. (158) <223> REP fragment <220> <221> DOMAIN &Lt; 222 > (159) .. (165) <223> Spacer fragment <220> <221> DOMAIN <222> (166). (263) <223> CT fragment <400> 2 Gly Pro Asn Ser Gly Gln Gly Gly Gly Gly Gly Gly Leu Gly Gln Gly Gly 1 5 10 15 Tyr Gly Gln Gly Ala Gla Ser Ser Ala Ala Ala Ala Ala Ala Ala             20 25 30 Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly         35 40 45 Gly Gly Gly Gly Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala     50 55 60 Ala Ala Ala Ala Gly Arg Gly Gly Gly Gly Gly Tyr Gly Gln Gly Ser 65 70 75 80 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala                 85 90 95 Ala Ala Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gln Ser Gln Gly             100 105 110 Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala         115 120 125 Ala Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gln Gly Gly Gly Gly Gly Tyr     130 135 140 Gly Gln Ser Ser Ala Ser Ser Ala Ser Ala Ser Ser Ser Ala Ser Ser 145 150 155 160 Val Ala Asn Ser Val Ser Ser Leu Ser Ser Ser Ser Ala Val Ser Arg                 165 170 175 Val Ser Ser Ala Val Ser Ser Leu Val Ser Asn Gly Gln Val Asn Met             180 185 190 Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile Ser Ser Ser Val Ser Ala         195 200 205 Ser Ala Pro Gly Ala Ser Gly Cys Glu Ale Leu Leu     210 215 220 Glu Val Ile Thr Ala Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly 225 230 235 240 Tyr Ile Asn Pro Ser Ala Val Asn Gln Ile Thr Asn Val Val Ala Asn                 245 250 255 Ala Met Ala Gln Val Met Gly             260 <210> 3 <211> 98 <212> PRT <213> Euprosthenops australis <400> 3 Ser Arg Ser Ser Ser Ser Ser Val Ser Ser Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn             20 25 30 Ile Ile Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala         35 40 45 Ser Gly Cys Glu Val Ile Val Gln Ala Leu Glu Val Ile Thr Ala     50 55 60 Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser 65 70 75 80 Ala Val Asn Gln Ile Thr Asn Val Ala Asn Ala Met Ala Gln Val                 85 90 95 Met Gly          <210> 4 <211> 100 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence derived from known MaSp1 and MaSp2 proteins <220> <221> MISC_FEATURE <222> (1) (71) <223> Sequence length present in known species variants <220> <221> VARIANT <222> (7) (7) <223> Glu <400> 4 Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln Ile Val Gly Gln Ser Val Ala Gln Ala                 85 90 95 Leu Gly Glu Phe             100 <210> 5 <211> 1110 <212> PRT <213> Euprosthenops australis <220> <221> REPEAT <222> (7). (19) <220> <221> REPEAT &Lt; 222 > (20) <220> <221> REPEAT &Lt; 222 > (43) .. (56) <220> <221> REPEAT &Lt; 222 > (57) <220> <221> REPEAT &Lt; 222 > (71) <220> <221> REPEAT (84). (106) <220> <221> REPEAT &Lt; 222 > (107) <220> <221> REPEAT &Lt; 222 > (121) <220> <221> REPEAT &Lt; 222 > (135) .. (147) <220> <221> REPEAT <222> (148). (170) <220> <221> REPEAT <222> (171). (183) <220> <221> REPEAT <222> (184). (197) <220> <221> REPEAT <222> (198). (211) <220> <221> REPEAT (222) (234) <220> <221> REPEAT <222> (235). (248) <220> <221> REPEAT <222> (249). (265) <220> <221> REPEAT <222> (266). (279) <220> <221> REPEAT (280). (293) <220> <221> REPEAT &Lt; 222 > (294) <220> <221> REPEAT &Lt; 222 > (307). (329) <220> <221> REPEAT &Lt; 222 > (330) <220> <221> REPEAT &Lt; 222 > (343) .. (356) <220> <221> REPEAT <222> (357). (370) <220> <221> REPEAT &Lt; 222 > (371) .. (393) <220> <221> REPEAT &Lt; 222 > (394) .. (406) <220> <221> REPEAT &Lt; 222 > (407) .. (420) <220> <221> REPEAT <222> (421). (434) <220> <221> REPEAT <222> (435). (457) <220> <221> REPEAT <222> (458). (470) <220> <221> REPEAT &Lt; 222 > (471) .. (488) <220> <221> REPEAT <222> (489). (502) <220> <221> REPEAT &Lt; 222 > (503) .. (516) <220> <221> REPEAT &Lt; 222 > (517) .. (529) <220> <221> REPEAT &Lt; 222 > (530) .. (552) <220> <221> REPEAT <222> (553). (566) <220> <221> REPEAT <222> (567). (580) <220> <221> REPEAT &Lt; 222 > (581) .. (594) <220> <221> REPEAT &Lt; 222 > (595) .. (617) <220> <221> REPEAT &Lt; 222 > (618). (630) <220> <221> REPEAT &Lt; 222 > (631) .. (647) <220> <221> REPEAT &Lt; 222 > (648) .. (661) <220> <221> REPEAT <222> (662). (675) <220> <221> REPEAT <222> (676). (688) <220> <221> REPEAT <222> (689). (711) <220> <221> REPEAT (712). (725) <220> <221> REPEAT (726). (739) <220> <221> REPEAT <222> (740). (752) <220> <221> REPEAT <222> (753). (775) <220> <221> REPEAT &Lt; 222 > (776) .. (789) <220> <221> REPEAT <222> (790). (803) <220> <221> REPEAT <222> (804). (816) <220> <221> REPEAT <222> (817). (839) <220> <221> REPEAT <222> (840). (853) <220> <221> REPEAT <222> (854). (867) <220> <221> REPEAT <222> (868). (880) <220> <221> REPEAT <222> (881). (903) <220> <221> REPEAT (904). (917) <220> <221> REPEAT <222> (918). (931) <220> <221> REPEAT <222> (932). (945) <220> <221> REPEAT <222> (946). (968) <220> <221> REPEAT <222> (969). (981) <220> <221> REPEAT <222> (982). (998) <220> <221> REPEAT &Lt; 222 > (999). (1013) <220> <221> REPEAT &Lt; 222 > (1014) ... (1027) <220> <221> REPEAT <222> (1028). (1042) <220> <221> REPEAT <222> (1043). (1059) <220> <221> REPEAT <222> (1060). (1073) <220> <221> REPEAT <222> (1074). (1092) <400> 5 Gly Gly Gly Aly Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 15 Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly             20 25 30 Gly Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala         35 40 45 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly     50 55 60 Gln Gly Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 Ala Ala Ser Gly Gly Gly Gly Gln Gln Gly Gln Gly Gly Gln Gly Gln                 85 90 95 Gly Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala             100 105 110 Ala Ala Ala Ala Ala Ala Gly         115 120 125 Gly Gly Gly Aly Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     130 135 140 Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala                 165 170 175 Ser Ala Ala Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln             180 185 190 Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala         195 200 205 Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly     210 215 220 Gly Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala 225 230 235 240 Ala Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly                 245 250 255 Arg Tyr Gly Gln Gly Ala Gla Ser Ser Ala Ala Ala Ala Ala Ala             260 265 270 Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly         275 280 285 Gly Ala Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     290 295 300 Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 305 310 315 320 Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala                 325 330 335 Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly             340 345 350 Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly         355 360 365 Ala Ala Gly Gly Gly Gly Gly Gly Gly Tly Gly Gly Gly Gly Gly Gly Gly     370 375 380 Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala 385 390 395 400 Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly                 405 410 415 Ala Aly Aly Ala Ala Aly Aly Aly Aly Aly Aly Aly             420 425 430 Ala Ala Gly Gly Gly Gly Gly Gly Gly Tly Gly Gly Gly Gly Gly Gly Gly         435 440 445 Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala     450 455 460 Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gin Gly Arg 465 470 475 480 Tyr Gly Gln Gly Ala Gla Ser Ser Ala Ala Ala Ala Ala Ala Ala                 485 490 495 Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly             500 505 510 Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala         515 520 525 Ser Gly Gln Gly Ser Gln Gly Gly Gly Gly Gly Gly Gln Gly Gln Gly Gly     530 535 540 Tyr Gly Gln Gly Ala Gla Ser Ser Ala Ala Ala Ala Ala Ala Ala 545 550 555 560 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly Gly Gly Gly                 565 570 575 Ala Aly Aly Ala Ala Aly Aly Aly Aly Aly Aly Aly             580 585 590 Ala Ala Gly Gly Gly Gly Gly Gly Gly Tly Gly Gly Gly Gly Gly Gly Gly         595 600 605 Gly Tyr Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala     610 615 620 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr 625 630 635 640 Gly Gln Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala                 645 650 655 Ala Ala Ala Ala Gly Arg Gly Gly Gly Gly Gly Tyr Gly Gln Gly Ser             660 665 670 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser         675 680 685 Gly Gln Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr     690 695 700 Gly Gln Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala 705 710 715 720 Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly                 725 730 735 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala             740 745 750 Gly Gln Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr         755 760 765 Gly Gln Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala     770 775 780 Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly 785 790 795 800 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala                 805 810 815 Gly Gln Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr             820 825 830 Gly Gln Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala         835 840 845 Ala Ala Ala Ala Gly Arg Gly Gly Gly Gly Gly Tyr Gly Gln Gly Ser     850 855 860 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ser 865 870 875 880 Gly Gln Gly Ser Gln Gly Gly Gly Gly Gly Gly Gln Gly Gln Gly Gly Gly Tyr                 885 890 895 Gly Gln Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala             900 905 910 Ala Ala Ala Ala Ser Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ala         915 920 925 Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     930 935 940 Ala Gly Gln Gly Gly Gln Gly Gly 945 950 955 960 Tyr Gly Gln Gly Ala Gla Ser Ser Ala Ala Ala Ala Ala Ala Ala                 965 970 975 Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly             980 985 990 Gly Gly Gly Gly Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala         995 1000 1005 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly     1010 1015 1020 Ser Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala     1025 1030 1035 Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Arg Gln     1040 1045 1050 Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala     1055 1060 1065 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly     1070 1075 1080 Gly Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala     1085 1090 1095 Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Ser Ser     1100 1105 1110 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence derived from internal repeats of Euprosthenops        australis MaSp1 <220> <221> VARIANT <222> (4) (4) <223> Ser <220> <221> VARIANT &Lt; 222 > (8) <223> Tyr <220> <221> VARIANT &Lt; 222 > (11) <223> Gln <400> 6 Gly Gln Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Tyr 1 5 10 15 Gly Gln Gly Ala Gly Ser Ser             20 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence derived from internal repeats of Euprosthenops        australis MaSp1 <220> <221> VARIANT &Lt; 222 > (9) <223> Arg <220> <221> VARIANT &Lt; 222 > (14) <223> Ser <220> <221> VARIANT &Lt; 222 > (16) <223> Gly <400> 7 Gly Gln Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Gly Gly Ala Gly Ser 1 5 10 15 Ser      <210> 8 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Consensus sequence derived from internal repeats of Euprosthenops        australis MaSp1 <220> <221> VARIANT <222> (2) (2) <223> Gln <220> <221> VARIANT <222> (6) <223> Arg <220> <221> VARIANT &Lt; 222 > (11) <223> Ser <220> <221> VARIANT &Lt; 222 > (11) <223> Val <400> 8 Gly Arg Gly Gly Gly Gly Gly Asn Gly Gly Gly Gly Ala Gly Gly Asn 1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> cell-binding peptide <220> <221> misc_feature <223> X = any amino acid other than Cys <220> <221> DISULFID <222> (1) <400> 9 Cys Xaa Xaa Arg Gly Asp Xaa Xaa Xaa Cys 1 5 10 <210> 10 <211> 5 <212> PRT <213> Homo sapiens <400> 10 Ile Lys Val Ala Val 1 5 <210> 11 <211> 5 <212> PRT <213> Homo sapiens <400> 11 Tyr Ile Gly Ser Arg 1 5 <210> 12 <211> 5 <212> PRT <213> Homo sapiens <400> 12 Glu Pro Asp Ile Met 1 5 <210> 13 <211> 5 <212> PRT <213> Homo sapiens <400> 13 Asn Lys Asp Ile Leu 1 5 <210> 14 <211> 5 <212> PRT <213> Homo sapiens <400> 14 Gly Arg Lys Arg Lys 1 5 <210> 15 <211> 15 <212> PRT <213> Homo sapiens <400> 15 Lys Tyr Gly Ala Ala Ser Ile Lys Val Ala Val Ser Ala Asp Arg 1 5 10 15 <210> 16 <211> 12 <212> PRT <213> Homo sapiens <400> 16 Asn Gly Glu Pro Arg Gly Asp Thr Tyr Arg Ala Tyr 1 5 10 <210> 17 <211> 11 <212> PRT <213> Homo sapiens <400> 17 Pro Gln Val Thr Arg Gly Asp Val Phe Thr Met 1 5 10 <210> 18 <211> 12 <212> PRT <213> Homo sapiens <400> 18 Ala Val Thr Gly Arg Gly Asp Ser Ser Ala Ser Ser 1 5 10 <210> 19 <211> 8 <212> PRT <213> Homo sapiens <400> 19 Thr Gly Arg Gly Asp Ser Pro Ala 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Modified from Homo sapiens <220> <221> DISULFID <222> (1) <400> 20 Cys Thr Gly Arg Gly Asp Ser Pro Ala Cys 1 5 10 <210> 21 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Widhe et al., 2013 <400> 21 Gly Pro Asn Ser Arg Gly Asp Ala Gly Ala Ala Ser 1 5 10 <210> 22 <211> 10 <212> PRT <213> Homo sapiens <400> 22 Val Thr Gly Arg Gly Asp Ser Pro Ala Ser 1 5 10 <210> 23 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> modified from Homo sapiens <400> 23 Ser Thr Gly Arg Gly Asp Ser Ser Ala Ser 1 5 10 <210> 24 <211> 813 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein <400> 24 ggtccgaatt cacgcggcga tgcaggagcg gctagcggtc aaggaggata tggtggacta 60 ggtcaaggag ggtatggaca aggtgcagga agttctgcag ccgctgccgc cgccgcagca 120 gccgccgcag caggtggaca aggtggacaa ggtcaaggag gatatggaca aggttcagga 180 ggttctgcag ccgccgccgc cgccgcagca gcagcagcag ctgcagcagc tggacgaggt 240 caaggaggat atggccaagg ttctggaggt aatgctgctg ccgcagccgc tgccgccgcc 300 gccgccgctg cagcagccgg acagggaggt caaggtggat atggtagaca aagccaaggt 360 gctggttccg ctgctgctgc tgctgctgct gctgccgctg ctgctgctgc aggatctgga 420 caaggtggat acggtggaca aggtcaagga ggttatggtc agagtagtgc ttctgcttca 480 gctgctgcgt cagctgctag tactgtagct aattcggtga gtcgcctctc atcgccttcc 540 gcagtatctc gagtttcttc agcagtttct agcttggttt caaatggtca agtgaatatg 600 gcagcgttac ctaatatcat ttccaacatt tcttcttctg tcagtgcatc tgctcctggt 660 gcttctggat gtgaggtcat agtgcaagct ctactcgaag tcatcactgc tcttgttcaa 720 atcgttagtt cttctagtgt tggatatatt aatccatctg ctgtgaacca aattactaat 780 gttgttgcta atgccatggc tcaagtaatg ggc 813 <210> 25 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 25 Gly Pro Asn Ser Arg Gly Asp Ala Gly Ala Ala Ser Gly Gln Gly Gly 1 5 10 15 Tyr Gly Gly Leu Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ser Ser             20 25 30 Ala Ala Ala Ala Ala Ala Ala Ala Ala Aly Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly         35 40 45 Gly Gln Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly Ser Ala Ala     50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asn Ala Ala Ala Ala                 85 90 95 Ala Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly             100 105 110 Gly Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Ala Ala Gly Ser Gly     130 135 140 Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser 145 150 155 160 Ala Ala Ala Ser Ala Ala Ser Thr Val Ala Asn Ser Ala Val Ser Ser Leu                 165 170 175 Ser Ser Pro Ser Ser Val Ser Ser Val             180 185 190 Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser         195 200 205 Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys     210 215 220 Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln 225 230 235 240 Ile Val Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val Asn                 245 250 255 Gln Ile Thr Asn Val Ala Asn Ala Met Ala Gln Val Met Gly             260 265 270 <210> 26 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein <400> 26 ggtccgaatt catgcacagg tcgtggtgat tctccggcgt gcggatccgc tagcggtcaa 60 ggaggatatg gtggactagg tcaaggaggg tatggacaag gtgcaggaag ttctgcagcc 120 gctgccgccg ccgcagcagc cgccgcagca ggtggacaag gtggacaagg tcaaggagga 180 tatggacaag gttcaggagg ttctgcagcc gccgccgccg ccgcagcagc agcagcagct 240 gcagcagctg gacgaggtca aggaggatat ggccaaggtt ctggaggtaa tgctgctgcc 300 gcagccgctg ccgccgccgc cgccgctgca gcagccggac agggaggtca aggtggatat 360 ggtagacaaa gccaaggtgc tggttccgct gctgctgctg ctgctgctgc tgccgctgct 420 gctgctgcag gatctggaca aggtggatac ggtggacaag gtcaaggagg ttatggtcag 480 agtagtgctt ctgcttcagc tgctgcgtca gctgctagta ctgtagctaa ttcggtgagt 540 cgcctctcat cgccttccgc agtatctcga gtttcttcag cagtttctag cttggtttca 600 aatggtcaag tgaatatggc agcgttacct aatatcattt ccaacatttc ttcttctgtc 660 agtgcatctg ctcctggtgc ttctggatgt gaggtcatag tgcaagctct actcgaagtc 720 atcactgctc ttgttcaaat cgttagttct tctagtgttg gatatattaa tccatctgct 780 gtgaaccaaa ttactaatgt tgttgctaat gccatggctc aagtaatggg c 831 <210> 27 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <220> <221> DISULFID &Lt; 222 > (5) <400> 27 Gly Pro Asn Ser Cys Thr Gly Arg Gly Asp Ser Pro Ala Cys Gly Ser 1 5 10 15 Ala Ser Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly Gly Gly Tyr Gly             20 25 30 Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala         35 40 45 Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gln Gly     50 55 60 Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly                 85 90 95 Asn Ala Aslan Ala Aslan Ala Aslan Ala Aslan             100 105 110 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala Gly         115 120 125 Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly     130 135 140 Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln 145 150 155 160 Ser Ser Ala Ser Ser Ala Ser Ala Ser Ala Ser Ser Val Ala                 165 170 175 Asn Ser Ser Ser Ser Leu Ser Ser Ser Ser Val Val Ser Ser Val Ser             180 185 190 Ser Ala Val Ser Ser Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala         195 200 205 Leu Pro Asn Ile Ile Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala     210 215 220 Pro Gly Ala Ser Gly Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val 225 230 235 240 Ile Thr Ala Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly Tyr Ile                 245 250 255 Asn Pro Ser Ala Val Asn Gln Ile Thr Asn Val Val Ala Asn Ala Met             260 265 270 Ala Gln Val Met Gly         275 <210> 28 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 28 Gly Ser Gly Asn Ser Gly Ile Gln Gly Gln Gly Gly Tyr Gly Gly Leu 1 5 10 15 Gly Gln Gly Gly Tly Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala             20 25 30 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly         35 40 45 Gly Gly Tyr Gly Gly Gly Gly Gly Gly Gly Ser     50 55 60 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly 65 70 75 80 Gly Gn Gly Gly Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala                 85 90 95 Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Arg             100 105 110 Gly Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Gly Gly Gly     130 135 140 Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala Ser Ser 145 150 155 160 Ala Ala Ser Thr Val Ala Asn Ser Ser Ser Ser Leu Ser Ser Ser Ser                 165 170 175 Ala Val Ser Ser Val Val Ser Ser Val Ser Ser Le Val Val Ser Ser Gn             180 185 190 Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile Ser Ser         195 200 205 Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys Glu Val Ile Val     210 215 220 Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln Ile Val Ser Ser 225 230 235 240 Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val Asn Gln Ile Thr Asn                 245 250 255 Val Val Ala Asn Ala Met Ala Gln Val Met Gly             260 265 <210> 29 <211> 267 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 29 Gly Ser Gly Asn Ser Gly Ile Gln Gly Gln Gly Gly Tyr Gly Gly Leu 1 5 10 15 Gly Gln Gly Gly Tly Gly Gly Gly Gly Aly Gly Ser Ser Ala Ala Ala Ala             20 25 30 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly         35 40 45 Gly Gly Tyr Gly Gly Gly Gly Gly Gly Gly Ser     50 55 60 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly 65 70 75 80 Gly Gn Gly Gly Gly Gly Asn Ala Ala Ala Ala Ala Ala Ala Ala                 85 90 95 Ala Ala Ala Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Tyr Gly Arg             100 105 110 Gly Asp Gly Gly Gly Aly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Aly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Gly Gly Gly     130 135 140 Gln Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala Ser Ala Ala Ser Ser 145 150 155 160 Ala Ala Ser Thr Val Ala Asn Ser Ser Ser Ser Leu Ser Ser Ser Ser                 165 170 175 Ala Val Ser Ser Val Val Ser Ser Val Ser Ser Le Val Val Ser Ser Gn             180 185 190 Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile Ser Asn Ile Ser Ser         195 200 205 Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly Cys Glu Val Ile Val     210 215 220 Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val Gln Ile Val Ser Ser 225 230 235 240 Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val Asn Gln Ile Thr Asn                 245 250 255 Val Val Ala Asn Ala Met Ala Gln Val Met Gly             260 265 <210> 30 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 30 Gly Pro Asn Ser Gly Arg Lys Arg Lys Ala Gly Ala Ala Ser Gly Gln 1 5 10 15 Gly Gly Tyr Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Gly Aly Gly             20 25 30 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln         35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala     50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg 65 70 75 80 Gly Gln Gly Gly Tly Gly Gly Gly Gly Gly Gly Gly Asn Ala Ala Ala Ala                 85 90 95 Ala Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly             100 105 110 Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly     130 135 140 Tyr Gly Gly Gln Gly Gly Gly Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala 145 150 155 160 Ser Ala Ala Ser Ser Ala Ser Ser Val Ala Asn Ser Ser Ser Ser                 165 170 175 Leu Ser Ser Ser Ser Val Ser Ser Val Ser Ser Ala Val Ser Ser             180 185 190 Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile         195 200 205 Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly     210 215 220 Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val 225 230 235 240 Gln Ile Val Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val                 245 250 255 Asn Gln Ile Thr Asn Val Ala Asn Ala Met Ala Gln Val Met Gly             260 265 270 <210> 31 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 31 Gly Pro Asn Ser Ile Lys Val Ala Val Ala Gly Ala Arg Ser Gly Gln 1 5 10 15 Gly Gly Tyr Gly Gly Gly Gly Gly Gly Gly Tyr Gly Gly Gly Aly Gly             20 25 30 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gln         35 40 45 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala     50 55 60 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Arg 65 70 75 80 Gly Gln Gly Gly Tly Gly Gly Gly Gly Gly Gly Gly Asn Ala Ala Ala Ala                 85 90 95 Ala Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly             100 105 110 Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala Gly Ser Ala Ala Ala Ala         115 120 125 Ala Ala Ala Ala Ala Ala Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Aly Gly     130 135 140 Tyr Gly Gly Gln Gly Gly Gly Gly Gly Tyr Gly Gln Ser Ser Ala Ser Ala 145 150 155 160 Ser Ala Ala Ser Ser Ala Ser Ser Val Ala Asn Ser Ser Ser Ser                 165 170 175 Leu Ser Ser Ser Ser Val Ser Ser Val Ser Ser Ala Val Ser Ser             180 185 190 Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn Ile Ile         195 200 205 Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala Ser Gly     210 215 220 Cys Glu Val Ile Val Gln Ala Leu Leu Glu Val Ile Thr Ala Leu Val 225 230 235 240 Gln Ile Val Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser Ala Val                 245 250 255 Asn Gln Ile Thr Asn Val Ala Asn Ala Met Ala Gln Val Met Gly             260 265 270 <210> 32 <211> 18 <212> PRT <213> Euprosthenops australis <400> 32 Ala Ser Ala Ser Ala Ala Ser Ala Ser Ser Val Ala Asn Ser 1 5 10 15 Val Ser          <210> 33 <211> 8 <212> PRT <213> Euprosthenops australis <400> 33 Ala Ser Ala Ala Ala Ala Ala Ala 1 5 <210> 34 <211> 8 <212> PRT <213> Euprosthenops australis <400> 34 Gly Ser Ala Met Gly Gln Gly Ser 1 5 <210> 35 <211> 5 <212> PRT <213> Euprosthenops australis <400> 35 Ser Ala Ser Ala Gly 1 5 <210> 36 <211> 100 <212> PRT <213> Euprosthenops sp <400> 36 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser             20 25 30 Thr Ile Ser Asn Val Ser Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln Leu Val Gly Gln Ser Val Tyr Gln Ala                 85 90 95 Leu Gly Glu Phe             100 <210> 37 <211> 98 <212> PRT <213> Euprosthenops australis <400> 37 Ser Arg Ser Ser Ser Ser Ser Val Ser Ser Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Asn Gly Gln Val Asn Met Ala Ala Leu Pro Asn             20 25 30 Ile Ile Ser Asn Ile Ser Ser Ser Val Ser Ala Ser Ala Pro Gly Ala         35 40 45 Ser Gly Cys Glu Val Ile Val Gln Ala Leu Glu Val Ile Thr Ala     50 55 60 Leu Val Gln Ile Val Ser Ser Ser Ser Val Gly Tyr Ile Asn Pro Ser 65 70 75 80 Ala Val Asn Gln Ile Thr Asn Val Ala Asn Ala Met Ala Gln Val                 85 90 95 Met Gly          <210> 38 <211> 99 <212> PRT <213> Argiope trifasciata <400> 38 Ser Arg Leu Ser Ser Gly Ala Ser Ser Val Val Ser Ser Ala Val 1 5 10 15 Thr Ser Leu Val Ser Ser Gly Gly Pro Thr Asn Ser Ala Ala Leu Ser             20 25 30 Asn Thr Ile Ser Asn Val Ser Ser Gln Ile Ser Ser Asn Pro Gly         35 40 45 Leu Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser     50 55 60 Ala Leu Val His Ile Leu Gly Ser Ala Asn Ile Gly Gln Val Asn Ser 65 70 75 80 Ser Gly Val Gly Arg Ser Ala Ser Ile Val Gly Gln Ser Ile Asn Gln                 85 90 95 Ala Phe Ser              <210> 39 <211> 89 <212> PRT <213> Cyrtophora moluccensis <400> 39 Ser His Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Ser Thr Asn Ser Ala Ala Leu Pro Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ale Thr Gln Ile Val                 85 <210> 40 <211> 98 <212> PRT <213> Latrodectus geometricus <400> 40 Ser Ala Leu Ala Ala Pro Ala Thr Ser Ala Arg Ile Ser Ser His Ala 1 5 10 15 Ser Thr Leu Leu Ser Asn Gly Pro Thr Asn Pro Ala Ser Ile Ser Asn             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Ile Ser Ser Asn Pro Gly Ala         35 40 45 Ser Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val Thr Ala     50 55 60 Leu Leu Thr Ile Ile Gly Ser Ser Asn Val Gly Asn Val Asn Tyr Asp 65 70 75 80 Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Soy                 85 90 95 Phe Val          <210> 41 <211> 98 <212> PRT <213> Latrodectus hesperus <400> 41 Ser Ala Leu Ser Ala Pro Ala Thr Ser Ala Arg Ile Ser Ser His Ala 1 5 10 15 Ser Ala Leu Leu Ser Ser Gly Pro Thr Asn Pro Ala Ser Ile Ser Asn             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Ile Ser Ser Asn Pro Gly Ala         35 40 45 Ser Ala Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val Thr Ala     50 55 60 Leu Leu Thr Ile Ile Gly Ser Ser Asn Ile Gly Ser Val Asn Tyr Asp 65 70 75 80 Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Soy                 85 90 95 Phe Gly          <210> 42 <211> 93 <212> PRT <213> Macrothele holsti <400> 42 Ser His Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Gly Gly Ser Thr Asn Ser Ala Ala Leu Pro Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asp Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln Ile Val Gly Gln Ser Ala                 85 90 <210> 43 <211> 98 <212> PRT <213> Nephila clavipes <400> 43 Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ala Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser             20 25 30 Thr Ile Ser Asn Val Ser Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile Gln Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln Ile Val Gly Gln Ser Val Tyr Gln Ala                 85 90 95 Leu Gly          <210> 44 <211> 89 <212> PRT <213> Nephila pilipes <400> 44 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ale Thr Gln Ile Val                 85 <210> 45 <211> 87 <212> PRT <213> Nephila madagascariensis <400> 45 Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ala Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser             20 25 30 Thr Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln                 85 <210> 46 <211> 87 <212> PRT <213> Nephila senegalensis <400> 46 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Ser             20 25 30 Thr Ile Ser Asn Val Ser Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ala Thr Gln                 85 <210> 47 <211> 89 <212> PRT <213> Octonoba varians <400> 47 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Ser Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Pro Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ale Thr Gln Ile Val                 85 <210> 48 <211> 89 <212> PRT <213> Psechrus sinensis <400> 48 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Asn Ser Ala Ala Leu Pro Asn             20 25 30 Thr Ile Ser Asn Val Val Ser Gln Ile Ser Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Ile His Ile Leu Gly Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ala Gly Gln Ale Thr Gln Ile Val                 85 <210> 49 <211> 88 <212> PRT <213> Tetragnatha kauaiensis <400> 49 Ser Leu Leu Ser Ser Ala Ser Asn Ala Arg Ile Ser Ser Ala Val 1 5 10 15 Ser Ala Leu Ala Ser Gly Ala Ala Ser Gly Pro Gly Tyr Leu Ser Ser             20 25 30 Val Ile Ser Asn Val Ser Ser Gln Val Ser Ser Asn Ser Gly Gly Leu         35 40 45 Val Gly Cys Asp Thr Leu Val Gln Ala Leu Glu Ala Ala Ala Ala     50 55 60 Leu Val His Val Leu Ala Ser Ser Ser Gly Gly Gin Val Asn Leu Asn 65 70 75 80 Thr Ala Gly Tyr Thr Ser Gln Leu                 85 <210> 50 <211> 88 <212> PRT <213> Tetragnatha versicolor <400> 50 Ser Ser Leu Ser Ser Ala Ser Asn Ala Arg Ile Ser Ser Ala Val 1 5 10 15 Ser Ala Leu Ala Ser Gly Gly Ala Ser Ser Gly Tyr Leu Ser Ser             20 25 30 Ile Ile Ser Asn Val Val Ser Gln Val Ser Ser Asn Asn Asp Gly Leu         35 40 45 Ser Gly Cys Asp Thr Val Val Gln Ala Leu Leu Glu Val Ala Ala Ala     50 55 60 Leu Val His Val Leu Ala Ser Ser Asn Ile Gly Gln Val Asn Leu Asn 65 70 75 80 Thr Ala Gly Tyr Thr Ser Gln Leu                 85 <210> 51 <211> 89 <212> PRT <213> Araneus bicentenarius <400> 51 Ser Ser Leu Ser Ser Ser Ala Ser Ser Ser Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Ser Gly Pro Thr Thr Pro Ala Ala Leu Ser Asn             20 25 30 Thr Ile Ser Ser Ala Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ser Ser Val Gly Gln Ile Asn Tyr Gly 65 70 75 80 Ala Ser Ala Gln Tyr Ala Gln Met Val                 85 <210> 52 <211> 97 <212> PRT <213> Argiope amoena <400> 52 Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val Ser 1 5 10 15 Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn Ala             20 25 30 Ile Gly Ser Ser Val Ser Gly Val Ser Ala Ser Asn Pro Gly Leu Pro         35 40 45 Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala Leu     50 55 60 Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ser Ala 65 70 75 80 Ser Ser Gln Tyr Ala Arg Leu Val Gly Gln Ser Ile Ala Gln Ala Leu                 85 90 95 Gly      <210> 53 <211> 82 <212> PRT <213> Argiope aurantia <400> 53 Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ala Leu Ser Asn             20 25 30 Ala Ile Ser Ser Val Ser Ser Gln Val Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Val Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ala 65 70 75 80 Ala Ser          <210> 54 <211> 98 <212> PRT <213> Argiope trifasciata <400> 54 Ser Arg Leu Ser Ser Pro Gln Ala Ser Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Thr Leu Val Ser Ser Gly Pro Thr Asn Pro Ala Ser Leu Ser Asn             20 25 30 Ala Ile Ser Ser Val Val Ser Gln Val Ser Ser Sern Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Ala 65 70 75 80 Ala Ser Ser Gln Tyr Ala Gln Leu Val Gly Gln Ser Leu Thr Gln Ala                 85 90 95 Leu Gly          <210> 55 <211> 89 <212> PRT <213> Gasteracantha mammosa <400> 55 Ser Ser Leu Ser Ser Pro Gln Ala Gly Ala Arg Val Ser Ser Ala Val 1 5 10 15 Ser Ala Leu Val Ala Ser Gly Pro Thr Ser Pro Ala Ala Val Ser Ser             20 25 30 Ala Ile Ser Asn Val Ala Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Val Ser Ala     50 55 60 Leu Val Ser Ile Leu Ser Ser Ala Ser Ile Gly Gln Ile Asn Tyr Gly 65 70 75 80 Ala Ser Gly Gln Tyr Ala Ala Met Ile                 85 <210> 56 <211> 90 <212> PRT <213> Latrodectus geometricus <400> 56 Ser Ala Leu Ser Ser Pro Thr Thr His Ala Arg Ile Ser Ser His Ala 1 5 10 15 Ser Thr Leu Leu Ser Ser Gly Pro Thr Asn Ser             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Val Ser Ala Ser Asn Pro Gly Ser         35 40 45 Ser Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Leu Ile Thr Ala     50 55 60 Leu Ile Ser Ile Val Asp Ser Ser Asn Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ser Gly Gln Tyr Ala Gln Met Val Gly                 85 90 <210> 57 <211> 98 <212> PRT <213> Latrodectus hesperus <400> 57 Ser Ala Leu Ser Ser Pro Thr Thr His Ala Arg Ile Ser Ser His Ala 1 5 10 15 Ser Thr Leu Leu Ser Ser Gly Pro Thr Asn Ala Ala Leu Ser Asn             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Val Ser Ala Ser Asn Pro Gly Ser         35 40 45 Ser Ser Cys Asp Val Leu Val Gln Ala Leu Leu Glu Ile Ile Thr Ala     50 55 60 Leu Ile Ser Ile Leu Asp Ser Ser Val Gly Gln Val Asn Tyr Gly 65 70 75 80 Ser Ser Gly Gln Tyr Ala Gln Ile Val Gly Gln Ser Ser Gln Gln Ala                 85 90 95 Met Gly          <210> 58 <211> 97 <212> PRT <213> Nephila clavipes <400> 58 Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala Arg Val Ala Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu Ser Ser             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile Val Ser Ala     50 55 60 Cys Val Thr Ile Leu Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ala Ala Ser Gln Phe Ala Gln Val Val Gly Gln Ser Val Leu Ser Ala                 85 90 95 Phe      <210> 59 <211> 82 <212> PRT <213> Nephila madagascariensis <400> 59 Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala Arg Val Ala Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu Ser Ser             20 25 30 Val Ile Ser Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Gln Ala Leu Leu Glu Ile Val Ser Ala     50 55 60 Cys Val Thr Ile Leu Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ala Ala          <210> 60 <211> 82 <212> PRT <213> Nephila senegalensis <220> <221> misc_feature &Lt; 222 > (35) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature &Lt; 222 > (56) .. (56) <223> Xaa can be any naturally occurring amino acid <400> 60 Ser Arg Leu Ala Ser Pro Asp Ser Gly Ala Arg Val Ala Ser Ala Val 1 5 10 15 Ser Asn Leu Val Ser Ser Gly Pro Thr Ser Ser Ala Ala Leu Ser Ser             20 25 30 Val Ile Xaa Asn Ala Val Ser Gln Ile Gly Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Ile Xaa Ala Leu Leu Glu Ile Val Ser Ala     50 55 60 Cys Val Thr Ile Leu Ser Ser Ser Ile Gly Gln Val Asn Tyr Gly 65 70 75 80 Ala Ala          <210> 61 <211> 71 <212> PRT <213> Dolomedes tenebrosus <400> 61 Ser Arg Leu Ser Ser Pro Glu Ala Ser Ser Val Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Asn Gly Gln Val Asn Val Asp Ala Leu Pro Ser             20 25 30 Ile Ile Ser Asn Leu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser I Ser         35 40 45 Ser Asp Cys Glu Val Leu Val Glu Val Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Val Gln Ile Val Cys Ser 65 70 <210> 62 <211> 97 <212> PRT <213> Dolomedes tenebrosus <400> 62 Ser Arg Leu Ser Ser Pro Gln Ala Ala Ser Arg Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Asn Gly Gln Val Asn Val Ala Leu Pro Ser             20 25 30 Ile Ile Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser I Ser         35 40 45 Ser Asp Cys Glu Val Leu Val Gln Val Leu Leu Glu Ile Val Ser Ala     50 55 60 Leu Val Gln Ile Val Ser Ser Ala Asn Val Gly Tyr Ile Asn Pro Glu 65 70 75 80 Ser Ale Ser Ale Ser Ale Ser Ale Ser Ale Ser Ale Ser Ale Ser Ale Ale Ale                 85 90 95 Gly      <210> 63 <211> 93 <212> PRT <213> Araneus diadematus <400> 63 Asn Arg Leu Ser Ser Ala Gly Ala Ala Ser Arg Val Val Ser Ser Asn Val 1 5 10 15 Ala Ala Ile Ala Ala Ala Glu Ala Ala Ala Leu Pro Asn Val Ile Ser             20 25 30 Asn Ile Tyr Ser Gly Val Ser Ser Glu Ser         35 40 45 Leu Ile Gln Ale Leu Leu Glu Val Ile Ser     50 55 60 Gly Ser Ala Ser Ile Gly Asn Val Ser Ser Val Gly Val Asn Ser Ala 65 70 75 80 Leu Asn Ala Val Gln Asn Ala Val Gly Ala Tyr Ala Gly                 85 90 <210> 64 <211> 98 <212> PRT <213> Araneus diadematus <400> 64 Ser Arg Ser Ser Ser Ser Ser Ala Val Ser Ser Ala Val 1 5 10 15 Ser Leu Val Ser Asn Gly Gly Pro Thr Ser Pro Ala Ala Leu Ser Ser             20 25 30 Ser Ile Ser Asn Val Val Ser Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Ile Leu Val Gln Ala Leu Leu Glu Ile Ile Ser Ala     50 55 60 Leu Val His Ile Leu Gly Ser Ala Asn Ile Gly Pro Val Asn Ser Ser 65 70 75 80 Ser Ala Gly Gln Ser Ala Ser Ile Val Gly Gln Ser Val Tyr Arg Ala                 85 90 95 Leu Ser          <210> 65 <211> 98 <212> PRT <213> Araneus diadematus <400> 65 Ser Arg Leu Ser Ser Pro Ala Ala Ser Ser Val Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Ser Gly Pro Thr Lys His Ala Ala Leu Ser Asn             20 25 30 Thr Ile Ser Ser Val Ser Val Ser Ser Val Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Val Leu Val Gln Ala Leu Leu Glu Val Val Ser Ala     50 55 60 Leu Val Ser Ile Leu Gly Ser Ser Ser Ile Gly Gln Ile Asn Tyr Gly 65 70 75 80 Ala Ser Ala Gln Tyr Thr Gln Met Val Gly Gln Ser Val Ala Gln Ala                 85 90 95 Leu Ala          <210> 66 <211> 94 <212> PRT <213> Araneus diadematus <400> 66 Ser Val Tyr Leu Arg Leu Gln Pro Arg Leu Glu Val Ser Ser Ala Val 1 5 10 15 Ser Ser Leu Val Ser Ser Gly Pro Thr Asn Gly Ala Ala Val Ser Gly             20 25 30 Ala Leu Asn Ser Leu Val Ser Gln Ile Ser Ala Ser Asn Pro Gly Leu         35 40 45 Ser Gly Cys Asp Ala Leu Val Gl Ala Leu Leu Glu Leu Val Ser Ala     50 55 60 Leu Val Ala Ile Leu Ser Ser Ala Ser Ile Gly Gln Val Asn Val Ser 65 70 75 80 Ser Val Ser Gln Ser Thr Gln Met Ile Ser Gln Ala Leu Ser                 85 90 <210> 67 <211> 10 <212> PRT <213> Homo sapiens <400> 67 Ser Thr Gly Arg Gly Asp Ser Pro Ala Val 1 5 10 <210> 68 <211> 93 <212> PRT <213> Araneus ventricosus <400> 68 Asn Arg Leu Ser Ser Ala Glu Ala Ala Ser Arg Val Ser Ser Asn Ile 1 5 10 15 Ala Ala Ile Ala Ser Gly Gly Ala Ser Ala Leu Pro Ser Val Ile Ser             20 25 30 Asn Ile Tyr Ser Gly Val Val Ala Ser Gly Val Ser Ser Asn Glu Ala         35 40 45 Leu Ile Gln Ala Leu Leu     50 55 60 Ser Ser Ala Ser Ile Gly Asn Val Ser Ser Val Gly Val Asp Ser Thr 65 70 75 80 Leu Asn Val Val Gln Asp Ser Val Gly Gln Tyr Val Gly                 85 90 <210> 69 <211> 272 <212> PRT <213> Artificial Sequence <220> <223> Fusion protein <400> 69 Gly Pro Asn Ser Cys Thr Gly Arg Gly Asp Ser Pro Ala Cys Gly Ser 1 5 10 15 Ala Ser Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gln Gly Gly Gly Tyr Gly             20 25 30 Gln Gly Ala Gly Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala         35 40 45 Ala Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gln Gly     50 55 60 Ser Gly Gly Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 Ala Ala Ala Gly Arg Gly Gln Gly Gly Tyr Gly Gln Gly Ser Gly Gly                 85 90 95 Asn Ala Aslan Ala Aslan Ala Aslan Ala Aslan             100 105 110 Gly Gln Gly Gly Gln Gly Gly Tyr Gly Arg Gln Ser Gln Gly Ala Gly         115 120 125 Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly     130 135 140 Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Gln Gly Gly Tyr Gly Gln 145 150 155 160 Ser Ser Ala Ser Ser Ala Ser Ser Ala                 165 170 175 Gly Ala Val Asn Arg Leu Ser Ser Ala Glu Ala Ala Ser Arg Val Ser             180 185 190 Ser Asn Ile Ala Ala Ile Ala Ser Gly         195 200 205 Val Ile Ser Asn Ile Tyr Ser Gly Val Val Ala Ser Gly Val Ser Ser     210 215 220 Asn Glu Ala Leu Ile Gln Ala Leu Leu Glu Leu Leu Ser Ala Leu Val 225 230 235 240 His Val Leu Ser Ser Ala Ser Ile Gly Asn Val Ser Ser Val Gly Val                 245 250 255 Asp Ser Thr Leu Asn Val Val Gln Asp Ser Val Gly Gln Tyr Val Gly             260 265 270

Claims (13)

As a method of culturing eukaryotic cells,
(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;
(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture;
(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells to form a support material for culturing eukaryotic cells; And
(d) maintaining eukaryotic cells in the support material under conditions suitable for cell culture. The method according to claim 1,
Wherein the macrostructure has a shape selected from fibers, foams, films, fiber meshes, capsules and nets, preferably fibers or foams. 3. The method according to claim 1 or 2,
Eukaryotic cells are selected from mammalian cells and are preferably selected from the group consisting of primary cells and cell lines such as endothelial cells, fibroblasts, keratinocytes, skeletal muscle satellite cells, Skeletal muscle myoblast, Schwann cell, pancreatic? -Cell, pancreatic islet cell, hepatocyte and glioma-forming cell; And stem cells such as mesenchymal stem cells; Or a combination of at least two different types of mammalian cells. 4. The method according to any one of claims 1 to 3,
Wherein the silk protein is fibroin, e.g., silk fibroin. 4. The method according to any one of claims 1 to 3,
Wherein the silk protein is a spider web protein. 6. The method of claim 5,
Spiderweb proteins comprise or consist of protein moieties REP and CT , wherein
REP is L (AG) _n L, L (AG) _n AL, and L (GA) _n L, and L (GA) _n GL 70 to about 300 repetitive fragment of an amino acid residue selected from the group consisting of, where
Wherein n is an integer from 2 to 10;
Each individual A segment is an amino acid sequence of 8 to 18 amino acid residues wherein 0 to 3 amino acid residues are not Ala and the remaining amino acid residues are Ala;
Wherein each individual G segment is an amino acid sequence of 12 to 30 amino acid residues wherein at least 40% of the amino acid residues are Gly; And
Each separate L segment is a linker amino acid sequence of from 0 to 30 amino acid residues; And
CT is a fragment of 70 to 120 amino acid residues having at least 70% identity to SEQ ID NO: 3 or SEQ ID NO: 68;
Wherein the selective cell-binding motif is arranged at the end of the spider protein, or between the moieties, or in any moiety, preferably at the end of the spider protein. 7. The method according to any one of claims 1 to 6,
The silk protein may be a cell-binding motif such as RGD, IKVAV, YIGSR, EPDIM, NKDIL, GRKRK, KYGAASIKVAVSADR, 15), NGEPRGDTYRAY (SEQ ID NO: 16), PQVTRGDVFTM (SEQ ID NO: 17), AVTGRGDSPASS (SEQ ID NO: 18), TGRGDSPA (SEQ ID NO: 19), CTGRGDSPAC (SEQ ID NO: 20) and FN _cc (SEQ ID NO: 9); Preferably a cell-binding motif selected from FN _cc , GRKRK, IKVAV, RGD and CTGRGDSPAC, more preferably from FN _cc and CTGRGDSPAC;
Wherein FN _cc is C ¹ X ¹ X ² RGDX ³ X ⁴ X ⁵ C ² ;
Wherein each of X ¹ , X ² , X ³ , X ^4, and X ⁵ is independently selected from natural amino acid residues other than cysteine; And C &lt; ^{1 &gt;} and C &lt; ² &gt; are connected through a disulfide bond. As a method for producing a cell culture product,
(i) a support material for culturing eukaryotic cells; And (ii) eukaryotic cells that grow integrally with the support material; Way
(a) providing an aqueous solution of a silk protein that can be assembled into a water-insoluble macrostructure, wherein the silk protein optionally comprises a cell-binding motif;
(b) preparing an aqueous mixture of a silk protein and a eukaryotic sample, wherein the silk protein remains dissolved in the aqueous mixture; And
(c) allowing the silk protein to assemble into a water-insoluble macrostructure in the presence of eukaryotic cells, thereby forming a support material for culturing eukaryotic cells
Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; 9. The method of claim 8,
The macrostructure is as defined in claim 2; And / or
Eukaryotic cells are as defined in the third; And / or
Wherein the silk protein is the same as defined in any one of claims 4 to 7. As a cell culture product
(i) a water insoluble macro structure of a silk protein that can be assembled into a water-insoluble macro structure, wherein the silk protein optionally comprises a cell-binding motif, a support material for culturing eukaryotic cells; And (ii) a cell culture product comprising eukaryotic cells that grow integrally with the support material. 11. The method of claim 10,
9. A cell culture product obtainable or obtainable by the method according to claim 8 or 9. Use of a silk protein capable of being assembled into a water insoluble macrostructure in the formation of a support material for culturing eukaryotic cells in the presence of cells, wherein the support material is a water insoluble macro structure of silk protein; Wherein the silk protein optionally contains a cell-binding motif. 13. The method of claim 12,
The macrostructure is as defined in claim 2; And / or
Eukaryotic cells are as defined in claim 3; And / or
Wherein the silk protein is as defined in any one of claims 4 to 7.

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